Robin M. Yates

Mycobacterium tuberculosis and the environment within the phagosome

Summary:u2002 Once across the barrier of the epithelium, macrophages constitute the primary defense against microbial invasion. For most microbes, the acidic, hydrolytically competent environment of the phagolysosome is sufficient to kill them. Despite our understanding of the trafficking events that regulate phagosome maturation, our appreciation of the lumenal environment within the phagosome is only now becoming elucidated through real‐time functional assays. The assays quantify pH change, phagosome/lysosome fusion, proteolysis, lipolysis, and β‐galactosidase activity. This information is particularly important for understanding pathogens that successfully parasitize the endosomal/lysosomal continuum. Mycobacterium tuberculosis infects macrophages through arresting the normal maturation process of the phagosome, retaining its vacuole at pH 6.4 with many of the characteristics of an early endosome. Current studies are focusing on the transcriptional response of the bacterium to the changing environment in the macrophage phagosome. Manipulation of these environmental cues, such as preventing the pH drop to pH 6.4 with concanamycin A, abrogates the majority of the transcriptional response in the bacterium, showing that pH is the dominant signal that the bacterium senses and responds to. These approaches represent our ongoing attempts to unravel the discourse that takes place between the pathogen and its host cell.

Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxer dogs.

ABSTRACT The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs (n = 13) and controls (n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli. Three of four GCB-associated E. coli isolates adhered to, invaded, and replicated within cultured epithelial cells. Invasion triggered a“ splash”-type response, was decreased by cytochalasin D, genistein, colchicine, and wortmannin, and paralleled the behavior of the Crohns disease-associated strain E. coli LF 82. GCB E. coli and LF 82 were diverse in serotype and overall genotype but similar in phylogeny (B2 and D), in virulence gene profiles (fyuA, irp1, irp2, chuA, fepC, ibeA, kpsMII, iss), in having a larger genome size than commensal E. coli, and in the presence of novel multilocus sequence types. We conclude that GCB is associated with selective intramucosal colonization by E. coli. E. coli strains associated with GCB and Crohns disease have an adherent and invasive phenotype and novel multilocus sequence types and resemble E. coli associated with extraintestinal disease in phylogeny and virulence gene profile.

The Kinetics of Phagosome Maturation as a Function of Phagosome/Lysosome Fusion and Acquisition of Hydrolytic Activity

Professional phagocytes function at the hinge of innate and acquired immune responses by internalizing particulate material that is digested and sampled within the phagosome of the cell. Despite intense interest, assays to measure phagosome maturation remain insensitive and few in number. In this current study, we describe three novel assays that quantify important biological properties of the phagosome as it matures. One assay exploits fluorescence resonance energy transfer to quantify mixing of phagocytosed particles carrying a donor fluor with an acceptor fluor loaded previously into the lysosomes as a fluid phase marker. Two additional assays describe the functional maturation of the phagosome as a hydrolytic compartment following the degradation of specifically designed peptide and triglyceride fluorogenic substrates.u2003The peptide substrate is preferentially cleaved by cysteine proteinases, and its degradation reflects proteinase delivery and activation within the acidifying phagosome. The fluorescence emission of the triglyceride analogue profiles the kinetics of triglyceride lipase activity within the phagosome. The fluorescence profiles of all three assays are modulated by known inhibitors of phagosome maturation, demonstrating the veracity, sensitivity and versatility of the assays.

Macrophage Activation Downregulates the Degradative Capacity of the Phagosome

The phagosome is key to most macrophage functions. It is the site of degradation of particulate material, of bacterial killing and the generation of peptides for antigen presentation. Despite its role at the fulcrum of the innate and acquired immune systems, little is known about the physiology of this organelle in activated macrophages. In this study, we utilize fluorometric techniques to characterize functional alterations in the lumenal environment of the maturing phagosome following stimulation of macrophages with interferon‐γ and/or lipopolysaccharide. In addition to modulating the kinetics of phagosomal acidification, activation results in a phagosome with diminished hydrolytic activities that varies markedly with the activation status of the cell. Differential levels of proteolytic, lipolytic and β‐galactosidase activities were observed in the phagosome but not in the total lysosomal extract, indicating selective delivery of enzymes to the developing phagosome. Despite the suppression of hydrolytic activities observed in early phagosomes, late phagosomes exhibit an enhanced and protracted accumulation of lysosomal cargo. The data are consistent with limiting proteolysis in the early phagosome to maximize epitope generation and antigen presentation while sequestering the degradative capacity in the late phagolysosome.

Intraphagosomal Measurement of the Magnitude and Duration of the Oxidative Burst

Generation of an oxidative burst within the phagosomes of neutrophils, dendritic cells and macrophages is an essential component of the innate immune system. To examine the kinetics of the oxidative burst in the macrophage phagosome, we developed two new assays using beads coated with oxidation‐sensitive fluorochromes. These assays permitted quantification and temporal resolution of the oxidative burst within the phagosome. The macrophage phagosomal oxidative burst is short lived, with oxidation of bead‐associated substrates reaching maximal activity within 30u2003min following phagocytosis. Additionally, the extent and rate of macrophage phagosomal substrate oxidation were subject to immunomodulation by activation with lipopolysaccharide and/or interferon‐γ.

Real-Time Spectrofluorometric Assays for the Lumenal Environment of the Maturing Phagosome

The ultimate goal of phagosomal maturation is the delivery of internalized, particulate cargo to acidic, hydrolytically competent compartments capable of mediating its degradation. Here we outline in detail three fluorometric techniques that allow the study of phagosomal maturation in macrophages by quantifying functionally important features of the lumenal environment of the developing phagosome in real time. The first assay utilizes a particle-restricted, pH-sensitive fluorochrome to measure the acidification of the phagosome. The second reports on the development of the proteolytic capacity of the phagosome by following the hydrolysis of a fluorogenic, generic proteinase substrate. The third quantifies the accumulation of lysosomal constituents within the phagosome by measuring the fluorescence resonance energy transfer (FRET) efficiency between a particle-restricted, donor fluor and a fluid phase acceptor fluor that had been chased previously into lysosomes. The assays are described as population-based methodologies utilizing a spectrofluorometer but, alternatively, can be adapted readily to confocal-based technologies for single phagosomal measurements.

TLR signalling and phagosome maturation: an alternative viewpoint.

In the February issue of Cellular Microbiology Blander expounds on the role of Toll-like receptors (TLRs) in intraphagosomal sensing and the modulation of the nascent phagosome (Blander, 2007a). The review extends the hypothesis of Medzhitov and Blander that the innate sensing machinery is capable of triggering an accelerated or ‘inducible’ maturation that results in a more hostile intraphagosomal environment (Blander and Medzhitov, 2004; 2006a). Attractive though this hypothesis of TLR-mediated phagosomal autonomy may be it is contradicted directly by data from our study that used real-time, kinetic analysis of the rates of phagosome acidification and phagosome–lysosome fusion in the presence and absence of stimulation by particle-associated TLR agonists (Yates and Russell, 2005; Yates et al., 2005). Given that these papers have been overlooked in recent reviews (Blander, 2007a,b), we feel it important to inform readers of the substantive data arguing that TLR stimulation does not impact on phagosome maturation. In our study we employed novel fluorometric assays that facilitate real-time measurement of pH through ratiometric fluorescence and phagosome–lysosome fusion through fluorescence resonance energy transfer to probe the kinetics of phagosome maturation (Yates and Russell, 2005; Yates et al., 2005). Initial experiments examined the rate of maturation of IgG beadand mannosylated beadcontaining phagosomes in the presence or absence of lipopolysaccharide (LPS) or Pam3Cys. The experiments were performed on both wild-type and TLR2and TLR4deficient macrophages. Although we could observe TLRmediated signalling in the presence of coupled TLR agonist and TLR, detected by p38 phosphorylation and a-IkB degradation, we could not observe differences in the rate or extent of phagosome maturation. In a recent review by Blander and Medzhitov the authors argue that the use of Fc receptors or the mannose receptor provides the ‘inducible’ signal without the need for stimulation of TLRs (Blander and Medzhitov, 2006a). Unfortunately the authors failed to acknowledge that in our study we then went on to examine the phagocytic processing of additional particles (Yates and Russell, 2005). We studied uptake of Staphylococcus aureus, one of the particles also used by Blander and Medzhitov, and employed phosphatidylserine-coated beads as a surrogate for the apoptotic cells to reproduce the ‘noninflammatory’ uptake pathway invoked in their earlier publication (Blander and Medzhitov, 2004). We looked at the kinetics of phagosome–lysosome fusion in phagosomes containing Staphylococcus aureus with and without absorbed LPS in both wild-type and TLR2-deficient macrophages. Once again although TLR-dependent signalling was detected under appropriate conditions, no differences in phagosome maturation was observed. Furthermore, the rate of maturation of phosphatidylserinecoated bead-containing phagosomes, mimicking apoptotic cells, also revealed no alterations in the kinetics of phagosome acidification despite addition of LPS or Pam3Cys to the particle. So why the discrepancy? One set of experiments in our study may hold the key. When we compared the rates of phagosome maturation in macrophages from MyD88deficient mice with those from parental strain controls we found that MyD88-deficient macrophages demonstrated diminished phagosome–lysosome fusion in the presence and absence of TLR agonist (Yates and Russell, 2005). Intriguingly however, LPS altered the kinetics and degree of phagosome–lysosome fusion in both wild-type and MyD88-deficient macrophages. This result has two important implications. First, MyD88-deficient macrophages have a phagosome maturation defect that is independent of the absence of short-term TLR-dependent signalling. Second, LPS has the capacity to modulate phagosome maturation independently of the action of TLR stimulation through MyD88. How do we interpret the data from Blander and Medzhitov in the context of our results? Analysis of the publication reporting the TLR-dependent modulation of Received 8 February, 2007; accepted 15 February, 2007. *For correspondence. E-mail dgr8@cornell.edu; Tel. (+1) 607 2533401; Fax (+1) 607 2534058. Cellular Microbiology (2007) 9(4), 849–850 doi:10.1111/j.1462-5822.2007.00920.x

Recording phagosome maturation through the real-time, spectrofluorometric measurement of hydrolytic activities.

The efficient degradation of internalized particulate matter is a principal objective of the macrophages phagosome. Assessment of the true hydrolytic capacity within the phagosomal lumen is often difficult as it is subject to many factors beyond recruitment of lysosomal hydrolases. Here we outline three assays that allow quantitative measurements of serine-cysteine protease, triglyceride lipase, and beta-galactosidase activities within the phagosomes of macrophages, in real time. The assays utilize ratio fluorometry between particle-associated fluorogenic substrates and calibration fluorochromes to yield internally controlled values that record rates of substrate hydrolysis. The methods described utilize a spectrofluorometer for fluorometric measurements from a population of macrophages. These assays, however, can be expanded to high-throughput or single cell formats. In addition, this approach can be applied to measure a wide variety of phagosomal hydrolytic properties with the design of suitable fluorogenic substrates.

Toll-like receptors and phagosome maturation.

To the editor: In a recent Perspective in Nature Immunology1, Blander and Medzhitov explored the function of Toll-like receptor (TLR) signaling in controlling the maturation of phagosomes in macrophages and dendritic cells. As the phagosome is the key organelle for the degradation of microbes and for the generation of bacterial peptides to be presented to lymphocytes, this issue is fundamental for understanding the innate-acquired immune interface. In their Perspective, Blander and Medzhitov discuss data from their published studies that found a key function for TLR signaling in the maturation of phagosomes. However, Blander and Medzhitov also revisit our paper in which we reported no influence of TLR signaling on phagosome maturation in macrophages2. Their comments, offering an alternative interpretation of our data, are based on the idea that some phagocytic receptors are ‘more equal than others’. In our paper, we used real-time, quantitative assays to measure the rates of acidification and phagosome-lysosome fusion phagosomes formed around silica beads bearing ligands (immunoglobulin G or mannosylated BSA), both with and without TLR agonists2,3. Blander and Medzhitov speculate that the beads we used, internalized by Fc receptors and mannose receptors, ‘maxed out’ those receptors and thus the rapid phagosome maturation program so that possible effects of additional TLR agonists added to those particles could not be discerned. That interpretation is proposed alongside descriptions of their own studies on phagosome cargos, including Escherichia coli, Salmonella typhimurium and Staphylococcus aureus, which they state are more relevant than immunoglobulin G or mannosylated BSA for the ‘nonopsonic’ uptake pathways they studied4. In their Perspective, Blander and Medzhitov did not mention that we studied real-time kinetics of maturation and that to exclude the possibility that Fc receptors and mannose receptors provided too ‘dominant’ a signal, we examined beads in the context of two conditions beyond use of immunoglobulin G and mannosylated BSA. We supplied wild-type and TLR2-deficient macrophages with fixed S. aureus, with and without lipopolysaccharide (LPS)2 and, as a ‘surrogate’ for apoptotic cells, we assessed phosphatidylserine-mediated uptake with lipid-coated beads in the presence and absence of the TLR agonists LPS and Pam3Cys (tripalmitoyl cysteinyl lipopeptide) 2. In neither of those series of experiments did we find any effect on the rate of phagosome maturation, despite manipulation of both the TLR agonist and the TLR receptor and verification of appropriate TLR activation. In summary, we found no effect of TLR signaling on the phagosomes formed around particles internalized through four distinct phagocytic routes. In contrast, we believe that the complexity of evaluating phagocytosis of E. coli, S. typhimurium and S. aureus, as used by Blander and Medzhitov, challenges any effort to establish the ‘receptor hierarchy’ proposed to explain the dichotomy between their results and ours. Moreover, rather than manipulating the TLR agonist makeup of their particles, Blander and Medzhitov relied soley on TLRdeficient phagocytes or phagocytes deficient in the MyD88 signaling adapter4. Two other results emerged from our study. First, we found that MyD88-deficient macrophages have less phagosome-lysosome fusion regardless of the identity of the internalized particles (which is perhaps a more likely explanation of the discrepancy between the results of our laboratories). Second, LPS-laden beads affect phagosome-lysosome fusion similarly in both wild-type and MyD88-deficient macrophages2. That finding, we believe, should raise concerns regarding the proposal by Blander and Medzhitov that LPS modulates dendritic cell phagosomes directly through TLR4 stimulation5, a proposal, we further believe, that would have benefited from verification by experiments with MyD88or TLR4-deficient cells. We therefore favor our conclusion that stimulation of TLRs by agonists present on the internalized particle does not affect the rate of phagosome maturation. David G Russell & Robin M Yates

DEVELOPMENT OF A NOVEL, CELL-BASED CHEMICAL SCREEN TO IDENTIFY INHIBITORS OF INTRAPHAGOSOMAL LIPOLYSIS IN MACROPHAGES

Macrophages play a central role in tissue homeostasis and the immune system. Their primary function is to internalize cellular debris and microorganisms for degradation within their phagosomes. In this context, their capacity to process and sequester lipids such as triacylglycerides and cholesteryl esters makes them key players in circulatory diseases, such as atheroclerosis. To discover new inhibitors of lipolytic processing within the phagosomal system of the macrophage, we have developed a novel, cell‐based assay suitable for high‐throughput screening. We employed particles carrying a fluorogenic triglyceride substrate and a calibration fluor to screen for inhibitors of phagosomal lipolysis. A panel of secondary assays were employed to discriminate between lipase inhibitors and compounds that perturbed general phagosomal trafficking events. This process enabled us to identify a new structural class of pyrazole‐methanone compounds that directly inhibit lysosomal and lipoprotein lipase activity.