Michel Rabinovitch

Professional and non-professional phagocytes: an introduction

Phagocytosis is the uptake by the cell of relatively large particles (>-OS pm) into vacuoles, by mechanisms that are clathrin independent and usually re- quire actin polymerization’ J. It is trig- gered by the interaction of certain mol- ecules on the particle surface with re- ceptors, lectins or other molecules on the phagocyte surface. Particles may be recognized directly by these receptors, but in many instances recognition is mediated by opsonins, such as anli- bodies, which coat the particles and are themselves recognized by specific receptorG. The vesicles containing the ingested material - phagosomes- ma- ture into phagolysosomes, which are acidic and rich in hydrolytic enzymes. In the Metazoa, phagocytosis underlies the uptake and degradation of microorganisms, damaged or senescent cells, and particulates such as pollutants, by specialized phago- cytic cells. Thus, phagocytasis is cen- tral to host defence against infective agents, and to tissue remodelling and inflammation. Paradoxically, phago- cytosis is also a common mechanism by which ‘ intracellular’ microorganisms invade host cells and thus avoid direct destruction by serum antibodies and complementor by cytotoxic celW. Phagocytosis is related to pino- cytosis, the process responsible for the cellular uptake of fluid, solutes, col- loids, macromolecules, virusesorsmall particles, and for bulk internalization of the plasma membrane. In contrast to phagocytosis, pinocytosis takes place constitutively, is usually clathrin dependent and generally does not require actin polymerization. The minimum particle size necessary to trigger phagocytosis or the maximum size for pinocytosis has not been defined, but size alone is probably only one of the features that deter- mine the way a particle is taken UP’ ,~. The molecular mechanisms of receptor-mediated pinocytosis are much better known than those of phagocytosis. One reason for this is that molecular ligands are usually quite specific, whereas complex par- ticles such as bacteria are often rec- ognized by several receptors, some of them incompletely characterized. In addition, whereas a wide range of cells can be used to study pinocytosis, work on phagocytosis has been primarily performed with polymorphonuclear granulocytes (PMNs), monocytes, pri- mary rmacrophage cultures or mono- cyte-macrophage-like cell lines; all these cells are difficult to transfect, dis- play several phagocytic receptors that can act cooperatively, and respond to

Autophagy induction favours the generation and maturation of the Coxiella -replicative vacuoles

Pathogens evolved mechanisms to invade host cells and to multiply in the cytosol or in compositionally and functionally customized membrane‐bound compartments. Coxiella burnetii, the agent of Q fever in man is a Gram‐negative γ‐proteobacterium which multiplies in large, acidified, hydrolase‐rich and fusogenic vacuoles with phagolysosomal‐like characteristics. We reported previously that C. burnetii phase II replicative compartments are labelled by LC3, a protein specifically localized to autophagic vesicles. We show here that autophagy in Chinese hamster ovary cells, induced by amino acid deprivation prior to infection with Coxiella increased the number of infected cells, the size of the vacuoles, and their bacterial load. Furthermore, overexpression of GFP‐LC3 or of GFP‐Rab24 – a protein also localized to autophagic vacuoles – likewise accelerated the development of Coxiella‐vacuoles at early times after infection. However, overexpression of mutants of those proteins that cannot be targeted to autophagosomes dramatically decreased the number and size of the vacuoles in the first hours of infection, although by 48 h the infection was similar to that of non‐transfected controls. Overall, the results suggest that transit through the autophagic pathway increases the infection with Coxiella by providing a niche more favourable to their initial survival and multiplication.

Coxiella burnetii Localizes in a Rab7-Labeled Compartment with Autophagic Characteristics

ABSTRACT The obligate intracellular bacterium Coxiella burnetii, the agent of Q fever in humans and of coxiellosis in other animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fuse homo- and heterotypically with other vesicles. To further characterize these vacuoles, HeLa cells were infected with C. burnetii phase II; 48 h later, bacteria-containing vacuoles were labeled by LysoTracker, a marker of acidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers of autophagic vacuoles. Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages in the autophagic process, each blocked Coxiella vacuole formation. These autophagosomal features suggest that Coxiella vacuoles interact with the autophagic pathway. The localization and role of wild-type and mutated Rab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the role of these small GTPases in the trafficking of C. burnetii phase II. Green fluorescent protein (GFP)-Rab5 and GFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy. Coxiella-containing large vacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas no colocalization was observed with the dominant-negative mutant Rab7T22N. The vacuoles were also decorated by GFP-Rab5Q79L but not by GFP-Rab5wt. These results suggest that Rab7 participates in the biogenesis of the parasitophorous vacuoles.

The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell

The etiologic agent of Q fever Coxiella burnetii, is an intracellular obligate parasite that develops large vacuoles with phagolysosomal characteristics, containing multiple replicating bacteria. We have previously shown that Phase II C. burnetii replicative vacuoles generated after 24–48 h post infection are decorated with the autophagic protein LC3. The aim of the present study was to examine, at earlier stages of infection, the distribution and roles of the small GTPases Rab5 and Rab7, markers of early and late endosomes respectively, as well as of the protein LC3 on C. burnetii trafficking. Our results indicate that: (i) Coxiella phagosomes (Cph) acquire the two Rab proteins sequentially during infection; (ii) overexpression of a dominant negative mutant form of Rab5, but not of Rab7, impaired Coxiella entry, whereas both Rab5 and Rab7 dominant negative mutants inhibited vacuole formation; (iii) Cph colocalized with the protein LC3 as early as 5 min after infection; acquisition of this protein appeared to be a bacterially driven process, because it was inhibited by the bacteriostatic antibiotic chloramphenicol and (iv) C. burnetii delayed the arrival of the typical lysosomal protease cathepsin D to the Cph, which delay is further increased by starvation‐induced autophagy. Based on our results we propose that C. burnetii transits through the normal endo/phagocytic pathway but actively interacts with autophagosomes at early times after infection. This intersection with the autophagic pathway delays fusion with the lysosomal compartment possibly favouring the intracellular differentiation and survival of the bacteria.

Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system.

Coxiella burnetii is an obligate intracellular pathogen that replicates in large endocytic vacuoles. Genomic sequence data indicate that 21 genes encoding products that are similar to components of the Legionella pneumophila Dot/Icm type IV secretion system are located on a contiguous 35 kb region of the Coxiella chromosome. It was found that several dot/icm genes were expressed by Coxiella during host cell infection and that dot/icm gene expression preceded the formation of large replicative vacuoles. To determine whether these genes encode a functional type IV secretion system, we have amplified the Coxiella dotB, icmQ, icmS and icmW genes and produced the encoded proteins in Legionella mutants in which the native copy of each gene had been deleted. The Coxiella dotB, icmS and icmW products restored dot/icm‐dependent growth of Legionella mutants in eukaryotic host cells. The Coxiella IcmQ protein and the Legionella IcmR protein did not interact, which could explain why the Coxiella icmQ gene was unable to restore growth to a Legionella icmQ mutant. Thus, Coxiella encodes functional components of a type IV secretion system expressed in vivo that is mechanistically related to the Legionella Dot/Icm apparatus. These studies suggest that a dot/icm‐related secretion system could play an important role in creating the specialized vacuole that supports Coxiella replication.

Nitric Oxide Partially Controls Coxiella burnetii Phase II Infection in Mouse Primary Macrophages

ABSTRACT In most primary or continuous cell cultures infected with the Q-fever agent Coxiella burnetii, bacteria are typically sheltered in phagolysosome-like, large replicative vacuoles (LRVs). We recently reported that only a small proportion of mouse peritoneal macrophages (PMΦ) infected with a nonvirulent, phase II strain of C. burnetii developed LRVs and that their relative bacterial load increased only slowly. In the majority of infected PMΦ, the bacteria were confined to the small vesicles. We show here that nitric oxide (NO) induced by the bacteria partially accounts for the restricted development of LRVs in primary macrophages. Thus, (i) PMΦ and bone marrow-derived macrophages (BMMΦ) challenged with phase II C. burnetii produced significant amounts of NO; (ii) the NO synthase inhibitors aminoguanidine and N-methyl-l-arginine reduced the production of NO and increased the frequency of LRVs (although the relative bacterial loads of individual LRVs did not change, the estimated loads per well increased appreciably); (iii) gamma interferon (IFN-γ) or the NO donor sodium nitroprusside, added to BMMΦ prior to or after infection, reduced the development and the relative bacterial loads of LRVs and lowered the yield of viable bacteria recovered from the cultures; and (iv) these effects of IFN-γ may not be entirely dependent on the production of NO since IFN-γ also controlled the infection in macrophages from inducible NO synthase knockout mice. It remains to be determined whether NO reduced the development of LRVs by acting directly on the bacteria; by acting on the traffic, fusion, or fission of cell vesicles; or by a combination of these mechanisms.

Cohabitation of Leishmania amazonensis and Coxiella burnetii

Intracellular pathogens customize the composition and function of the vacuoles they occupy, and can arrest or distort vacuolar maturation. In doubly infected cells, vacuoles that contain two different parasites can be used to test for exclusionary mechanisms, for expression of vacuolar phenotypes that permit or restrict fusion, and for the survival of pathogens targeted to an unusual cellular compartment.

Infection of Vero cells with Coxiella burnetii phase II: relative intracellular bacterial load and distribution estimated by confocal laser scanning microscopy and morphometry.

Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is an intracellular pathogen not yet grown axenically. Confocal laser fluorescence microscopy and morphometry were used to measure relative C. burnetii phase II loads and their intracellular distribution in aldehyde fixed and DAPI stained Vero cell monolayers. The fluorescence of single horizontal optical sections provided useful information on relative loads of bacteria in cells and vacuoles. The relative density of the bacteria in the vacuoles was inferred from ratios of fluorescence to vacuolar section areas. Relative bacterial loads, bacterial densities and section areas of large vacuoles increased exponentially between days 2 and 4 of the infection of gamma-irradiated host cells, stabilized between days 4 and 6, and decreased thereafter. Estimated minimum doubling times were higher for the overall complement of the intracellular organisms (about 12 h) than for bacteria that were confined to larger vacuoles (about 10 h).

Coinfection of fibroblasts with Coxiella burnetti and Toxoplasma gondii: to each their own

Intracellular pathogens have evolved distinct strategies to subvert host cell defenses. At diametrically opposed ends of the spectrum with regard to the host endosomal/lysosomal defenses are the obligate intracellular protozoan Toxoplasma gondii and the bacterium Coxiella burnetti. While the intracellular replication of T. gondii requires complete avoidance of the host endocytic cascade, C. burnetti actively subverts it. This results in these organisms establishing and growing in very different vacuolar compartments. In this study we examined the potential interaction between these distinct compartments following coinfection of mammalian fibroblasts. When present within the same cell, these organisms exhibit minimal interaction with each other. Colocalization of T. gondii and C. burnetti within the same vacuole occurs at a low frequency in doubly infected cells. In such instances only one of the organisms appears to be replication competent, emphasizing the different requirements for survival and/or intracellular growth. The potential basis for both the lack of interaction between these distinct pathogen-containing compartments, and the mechanisms to address their low frequency of colocalization are discussed in the context of our understanding of the biology of the organisms and membrane traffic in eukaryotic cells.

Phagocytosis of Apoptotic Cells Increases the Susceptibility of Macrophages to Infection with Coxiella burnetii Phase II through Down-Modulation of Nitric Oxide Production

ABSTRACT Coxiella burnetii, the agent of Q fever in humans and coxiellosis in other mammals, is an obligate intracellular bacterium which is sheltered and multiplies within typically large phagolysosome-like replicative vacuoles (LRVs). We have previously shown that, compared with fibroblasts, mouse resident peritoneal macrophages control the development of LRVs and bacterial multiplication within these vacuoles. Earlier experiments with the nitric oxide (NO) synthase inhibitor aminoguanidine (AG) revealed that the control is exerted by NO induced by the bacteria. We report here that phagocytosis of apoptotic-like, but not of aldehyde-killed, lymphocytes by the macrophages reduced the production of NO induced by the bacteria and increased the development of LRVs and, therefore, the total bacterial load in the cultures. Experiments with macrophages from mice deficient for inducible NO synthase (iNOS−/−) confirmed the involvement of NO in the control of infection, since neither apoptotic lymphocytes nor AG affected the development of LRVs in these phagocytes. Since macrophages are important for the clearance of apoptotic bodies and C. burnetii is able to induce apoptosis in human monocytes, the phenomenon shown here may be biologically relevant to the development of Q fever and coxiellosis.