Daniel L. Clemens

The Mannose Receptor Delivers Lipoglycan Antigens to Endosomes for Presentation to T Cells by CD1b Molecules

We have characterized the CD1b-mediated presentation pathway for the mycobacterial lipoglycan lipoarabinomannan (LAM) in monocyte-derived antigen-presenting cells. The macrophage mannose receptor (MR) was responsible for uptake of LAM. Antagonism of MR function inhibited both the internalization of LAM and the presentation of this antigen to LAM-reactive T cells. Intracellular MRs were most abundant in early endosomes, but they also were located in the compartment for MHC class II antigen loading (MIIC). Internalized LAM was transported to late endosomes, lysosomes, and MIICs. MRs colocalized with CD1b molecules, suggesting that the MR could deliver LAM to late endosomes for loading onto CD1b. LAM and CD1b colocalized in organelles that may be sites of lipoglycan antigen loading. This pathway links recognition of microbial antigens by a receptor of the innate immune system to the induction of adaptive T cell responses.

Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

ABSTRACT Francisella tularensis, the agent of tularemia, is an intracellular pathogen, but little is known about the compartment in which it resides in human macrophages. We have examined the interaction of a recent virulent clinical isolate of F. tularensis subsp. tularensis and the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy. We assessed the maturation of the F. tularensis phagosome by examining its acquisition of the lysosome-associated membrane glycoproteins (LAMPs) CD63 and LAMP1 and the acid hydrolase cathepsin D. Two to four hours after infection, vacuoles containing live F. tularensis cells acquired abundant staining for LAMPs but little or no staining for cathepsin D. However, after 4 h, the colocalization of LAMPs with live F. tularensis organisms declined dramatically. In contrast, vacuoles containing formalin-killed bacteria exhibited intense staining for all of these late endosomal/lysosomal markers at all time points examined (1 to 16 h). We examined the pH of the vacuoles 3 to 4 h after infection by quantitative immunogold staining and by fluorescence staining for lysosomotropic agents. Whereas phagosomes containing killed bacteria stained intensely for these agents, indicating a marked acidification of the phagosomes (pH 5.5), phagosomes containing live F. tularensis did not concentrate these markers and thus were not appreciably acidified (pH 6.7). An ultrastructural analysis of the F. tularensis compartment revealed that during the first 4 h after uptake, the majority of F. tularensis bacteria reside within phagosomes with identifiable membranes. The cytoplasmic side of the membranes of ∼50% of these phagosomes was coated with densely staining fibrils of ∼30 nm in length. In many cases, these coated phagosomal membranes appeared to bud, vesiculate, and fragment. By 8 h after infection, the majority of live F. tularensis bacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm. These results indicate that F. tularensis initially enters a nonacidified phagosome with LAMPs but without cathepsin D and that the phagosomal membrane subsequently becomes morphologically disrupted, allowing the bacteria to gain direct access to the macrophagic cytoplasm. The capacity of F. tularensis to alter the maturation of its phagosome and to enter the cytoplasm is likely an important element of its capacity to parasitize macrophages and has major implications for vaccine development.

Francisella tularensis enters macrophages via a novel process involving pseudopod loops

ABSTRACT Intracellular bacterial pathogens employ a variety of strategies to invade their eukaryotic host cells. From an ultrastructural standpoint, the processes that bacteria employ to invade their host cells include conventional phagocytosis, coiling phagocytosis, and ruffling/triggered macropinocytosis. In this paper, we describe a novel process by which Francisella tularensis, the agent of tularemia, enters host macrophages. F. tularensis is a remarkably infectious facultative intracellular bacterial parasite—as few as 10 bacteria can cause life-threatening disease in humans. However, the ultrastructure of its uptake and the receptor mechanisms that mediate its uptake have not been reported previously. We have used fluorescence microscopy and electron microscopy to examine the adherence and uptake of a virulent recent clinical isolate of F. tularensis, subspecies tularensis, and the live vaccine strain (LVS), subspecies holarctica, by human macrophages. We show here that both strains of F. tularensis enter human macrophages by a novel process of engulfment within asymmetric, spacious pseudopod loops, a process that differs ultrastructurally from all previously described uptake mechanisms. We demonstrate also that adherence and uptake of F. tularensis by macrophages is strongly dependent upon complement receptors and upon serum with intact complement factor C3 and that uptake requires actin microfilaments. These findings have significant implications for understanding the intracellular biology and virulence of this extremely infectious pathogen.

Characterization of the Mycobacterium tuberculosis phagosome

Mycobacterium tuberculosis parasitizes host macrophages and resides in a phagosome that does not fuse with lysosomes and that resists acidification. Compared with phagosomes containing inert particles, the M. tuberculosis phagosome exhibits maturational arrest: plasma membrane proteins and early endocytic markers persist and there is limited acquisition of lysosomal markers.

Mycobacterium tuberculosis and Legionella pneumophila Phagosomes Exhibit Arrested Maturation despite Acquisition of Rab7

ABSTRACT Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Phagosomes containing inert particles have also been shown to transiently acquire Rab7 as they mature. Disruption in the pathway prior to the acquisition of Rab7 has been suggested as playing a role in the altered maturation of Mycobacterium bovis BCG phagosomes. As a first step to determine whether disruption in the delivery or function of Rab7 could play a role in the altered maturation of Legionella pneumophila and M. tuberculosis phagosomes, we have examined the distribution of wild-type Rab7 and the GTPase-deficient, constitutively active mutant form of Rab7 in HeLa cells infected withL. pneumophila or M. tuberculosis. We have found that the majority of L. pneumophila and M. tuberculosis phagosomes acquire relatively abundant staining for Rab7 and for the constitutively active mutant Rab7 in HeLa cells that overexpress these proteins. Nevertheless, despite acquisition of wild-type or constitutively active Rab7, both the L. pneumophila and the M. tuberculosis phagosomes continue to exhibit altered maturation as manifested by a failure to acquire lysosome-associated membrane glycoprotein 1. These results demonstrate that L. pneumophila and M. tuberculosis phagosomes have receptors for Rab7 and that the altered maturation of these phagosomes is not due to a failure to acquire Rab7.

Atomic Structure of T6SS Reveals Interlaced Array Essential to Function.

Type VI secretion systems (T6SSs) are newly identified contractile nanomachines that translocate effector proteins across bacterial membranes. The Francisella pathogenicity island, required for bacterial phagosome escape, intracellular replication, and virulence, was presumed to encode a T6SS-like apparatus. Here, we experimentally confirm the identity of this T6SS and, by cryo electron microscopy (cryoEM), show the structure of its post-contraction sheath at 3.7 Å resolution. We demonstrate the assembly of this T6SS by IglA/IglB and secretion of its putative effector proteins in response to environmental stimuli. The sheath has a quaternary structure with handedness opposite that of contracted sheath of T4 phage tail and is organized in an interlaced two-dimensional array by means of β sheet augmentation. By structure-based mutagenesis, we show that this interlacing is essential to secretion, phagosomal escape, and intracellular replication. Our atomic model of the T6SS will facilitate design of drugs targeting this highly prevalent secretion apparatus.

Uptake and intracellular fate of Francisella tularensis in human macrophages.

Abstract:  Francisella tularensis is an intracellular pathogen that survives and multiplies within host mononuclear phagocytes. We have shown that uptake of the bacterium by human macrophages occurs by a novel process, “looping phagocytosis,” in which the bacterium is engulfed in a spacious, asymmetric, pseudopod loop. Whereas looping phagocytosis is resistant to treatment of the F. tularensis with formalin, proteases, or heat, the process is abolished by oxidation of the bacterial carbohydrates with periodate, suggesting a role for preformed surface carbohydrate molecules in triggering looping phagocytosis. Following uptake, F. tularensis initially resides in a spacious vacuole at the periphery of the cell, but this vacuole rapidly shrinks in size. The nascent F. tularensis vacuole transiently acquires early endosomal markers, but subsequently exhibits an arrested maturation, manifest by only limited amounts of lysosome‐associated membrane glycoproteins (consistent with limited interaction with late endosomes), nonfusion with lysosomes, and minimal acidification. In ultrastructural studies, we have observed that the F. tularensis phagosome displays a novel feature in that many of the phagosomes acquire an electron dense fibrillar coat. This fibrillar coat forms blebs and vesicles, and with time, is seen to be fragmented and disrupted. With increasing time after infection, increasing numbers of the F. tularensis are found free in the macrophage cytoplasm, such that by 14 h after infection, less than 15% of the bacteria are surrounded by any discernible phagosomal membrane. Further research is needed to determine the mechanisms underlying looping phagocytosis, and the maturational arrest, fibrillar coat formation, and disruption of the phagosome.

Targeted Intracellular Delivery of Antituberculosis Drugs to Mycobacterium tuberculosis-Infected Macrophages via Functionalized Mesoporous Silica Nanoparticles

ABSTRACT Delivery of antituberculosis drugs by nanoparticles offers potential advantages over free drug, including the potential to target specifically the tissues and cells that are infected by Mycobacterium tuberculosis, thereby simultaneously increasing therapeutic efficacy and decreasing systemic toxicity, and the capacity for prolonged release of drug, thereby allowing less-frequent dosing. We have employed mesoporous silica nanoparticle (MSNP) drug delivery systems either equipped with a polyethyleneimine (PEI) coating to release rifampin or equipped with cyclodextrin-based pH-operated valves that open only at acidic pH to release isoniazid (INH) into M. tuberculosis-infected macrophages. The MSNP are internalized efficiently by human macrophages, traffic to acidified endosomes, and release high concentrations of antituberculosis drugs intracellularly. PEI-coated MSNP show much greater loading of rifampin than uncoated MSNP and much greater efficacy against M. tuberculosis-infected macrophages. MSNP were devoid of cytotoxicity at the particle doses employed for drug delivery. Similarly, we have demonstrated that the isoniazid delivered by MSNP equipped with pH-operated nanovalves kill M. tuberculosis within macrophages significantly more effectively than an equivalent amount of free drug. These data demonstrate that MSNP provide a versatile platform that can be functionalized to optimize the loading and intracellular release of specific drugs for the treatment of tuberculosis.

Francisella tularensis Phagosomal Escape Does Not Require Acidification of the Phagosome

ABSTRACT Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by “F. tularensis subsp. novicida,” indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.

Massively parallel delivery of large cargo into mammalian cells with light pulses

We report a high-throughput platform for delivering large cargo elements into 100,000 cells in 1 min. Our biophotonic laser-assisted surgery tool (BLAST) generates an array of microcavitation bubbles that explode in response to laser pulsing, forming pores in adjacent cell membranes through which cargo is gently driven by pressurized flow. The platform delivers large items including bacteria, enzymes, antibodies and nanoparticles into diverse cell types with high efficiency and cell viability. We used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol.