Large Parasitophorous Vacuole Research Articles

Where is the EXIT? Phenotypic screens for new egress factors in apicomplexan parasites.

Apicomplexans, such as Plasmodium and Toxoplasma are obligate intracellular parasites that invade, replicate and finally EXIT their host cell. During replication within a parasitophorous vacuole (PV), the parasites establish an extensive F-actin-containing network that connects individual parasites and is required for material exchange, recycling and the final steps of daughter cell assembly. After multiple rounds of replication, the parasites exit the host cell involving multiple signalling cascades, disassembly of the network, secretion of microneme proteins and activation of the acto-myosin motor. Blocking the host cell EXIT process leads to the formation of large PVs, making the screening for genes involved in exiting the cell relatively straightforward. Given that apicomplexans are highly diverse from other eukaryotes, approximately 30% of all genes are annotated as hypothetical, some apicomplexan-specific factors are likely to be critical during EXIT. This motivated several labs to design and perform forward genetic and phenotypic screens using various approaches, such as random insertion mutagenesis, temperature-sensitive mutants and, more recently, CRISPR/Cas9-mediated targeted editing and conditional mutagenesis. Here we will provide an overview of the technological developments over recent years and the most successful stories that led to the identification of new critical factors in Toxoplasma gondii.

Causative Agents of American Tegumentary Leishmaniasis Are Able to Infect 3T3-L1 Adipocytes In Vitro

Although macrophages have long been considered key players in the course of Leishmania infections, other non-professional phagocytes have lately been shown to maintain low levels of the parasite in safe intracellular niches. Recently, it was demonstrated that the adipose tissue is capable of harboring Old World L. (L.) infantum in mice. However, there is no evidence of experimental adipocyte infection with New World Leishmania species so far. In addition, it was not known whether adipocytes would be permissive for formation of the unique, large and communal parasitophorous vacuoles that are typical of L. (L.) amazonensis in macrophages. Here we evaluated the ability of L. (L.) amazonensis and L. (V.) braziliensis promastigotes and amastigotes to infect 3T3-L1 fibroblast-derived adipocytes (3T3-Ad) using light and transmission electron microscopy. Our results indicate that amastigotes and promastigotes of both species were capable of infecting and surviving inside pre- and fully differentiated 3T3-Ad for up to 144 h. Importantly, L. (L.) amazonensis amastigotes resided in large communal parasitophorous vacuoles in pre-adipocytes, which appeared to be compressed between large lipid droplets in mature adipocytes. In parallel, individual L. (V.) braziliensis amastigotes were detected in single vacuoles 144 h post-infection. We conclude that 3T3-Ad may constitute an environment that supports low loads of viable parasites perhaps contributing to parasite maintenance, since amastigotes of both species recovered from these cells differentiated into replicative promastigotes. Our findings shed light on the potential of a new host cell model that can be relevant to the persistence of New World Leishmania species.

Isolation of intact Leishmania amazonensis large parasitophorous vacuoles from infected macrophages by density gradient fractionation

As the causative agent of hard-to-treat diffuse cutaneous leishmaniasis, Leishmania (L.) amazonensis persists in the host organism sheltered within large Parasitophorous Vacuoles (PVs) formed mainly in macrophages. In the present study, I present a simple and efficient method for L. amazonensis PV isolation. Isolated PVs are intact as demonstrated by the conservation of lysosomal probes loaded into PVs before the procedure. The method is useful for studies aiming at a complete and accurate molecular profile of these structures, to better understand the biogenesis of this pathogen-containing vacuole and its implication in parasite persistence and in leishmaniasis pathogenesis.

Dual Host-Intracellular Parasite Transcriptome of Enucleated Cells Hosting Leishmania amazonensis: Control of Half-Life of Host Cell Transcripts by the Parasite.

Enucleated cells or cytoplasts (cells whose nucleus is removed in vitro) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis, we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus. Dual RNA sequencing of both host cytoplast and intracellular parasite transcripts identified host transcripts that are more preserved or degraded upon interaction with parasites and also parasite genes that are differentially expressed when hosted by nucleated or enucleated cells. Cytoplasts are suitable host cells, which persist in culture for more than 72 h and display functional enrichment of transcripts related to mitochondrial functions and mRNA translation. Crosstalk between nucleated host de novo gene expression in response to intracellular parasitism and the parasite gene expression to counteract or benefit from these host responses induces a parasite transcriptional profile favoring parasite multiplication and aerobic respiration, and a host-parasite transcriptional landscape enriched in host cell metabolic functions related to NAD, fatty acid, and glycolytic metabolism. Conversely, interruption of host nucleus-parasite cross talk by infection of enucleated cells generates a host-parasite transcriptional landscape in which cytoplast transcripts are enriched in phagolysosome-related pathway, prosurvival, and SerpinB-mediated immunomodulation. In addition, predictive in silico analyses indicated that parasite transcript products secreted within cytoplasts interact with host transcript products conserving the host V-ATPase proton translocation function and glutamine/proline metabolism. The collective evidence indicates parasite-mediated control of host cell transcripts half-life that is beneficial to parasite intracellular multiplication and escape from host immune responses. These findings will contribute to improved drug targeting and serve as database for L. amazonensis-host cell interactions.

Light Microscopic Study of Caryospora sp. (Apicomplexa: Eimeriidae) from Savigny’s Agama Trapelus savignii (Reptilia: Agamidae) in Egypt

Developmental stages of a suggested new speciesof CaryosporaLeger, 1904 were described from the agamid lizards Trapelus savigniicaptured during summer 2019 from the Eastern Desert in Egypt. Overall recorded prevalence is 11.65% (12/103) and sporulation is endogenous. Sporulated oocysts are subspherical (70%) to spherical (30%) measuring 22.29–35.74 ×20.91–32.89 μm (length × width). The oocyst wall is bilayered, smooth, and measures 1.46–2.00 μm. Distinctive features of most oocysts include the presence of a sutured edge-like extension on one side of the oocyst wall and the ability of sporocyst contents to form a condensed curved mass within the sporocyst cavity. One or rarely two polar granules are present. Micropyle and oocyst residuum are absent. Sporocysts are ovoid to ellipsoidal measuring 17.17–22.76 × 12.86–16.76 μm. The morphometric descriptions and measurements of Stieda, sub-Stieda bodies, and sporocyst residuum are determined. Sporozoites are sausage-shaped and measure 5.69–8.59 × 2.08–3.29 μm. Merogonic stages and microgamonts occur mainly in the enterocytes of the small intestine. Endogenous stages are seen mostly with clear large parasitophorous vacuoles. Mature meronts measure 3.95–7.60 × 3.37–7.24μm and yield 3–11 merozoites with the formation of a residual body.Merozoites are banana-shaped measuring 2.51–3.29 × 0.72–1.20μm. A microgamont is noticed measuring in 9.53 × 8.42 μm.

Gametogony of Eimeria macusaniensis Guerrero, Hernandez, Bazalar and Alva, 1971 in llama (Lama glama).

Camelids (llama, alpaca, vicunãs, guanacos) are important for the economy of South America and Eimeria infections are an important cause of mortality in camelids. Of the six species of Eimeria in camelids, Eimeria macusaniensis, considered the most pathogenic, is distinctive; its oocysts are the largest among all Eimeria species in animals, its prepatent period is more than 1 month, and its oocysts have been found in mummies from prehistoric times. Although, E. macusaniensis gametogonic stages are found associated with enteritis in naturally infected camelids, the schizogonic stages are unknown and clinical disease has been reported in some camelids with no oocysts in feces. Described herein are morphological details of gametogonic development and oocyst formation of E. macusaniensis in a naturally infected llama (Lama lama), solely infected with this parasite. Microgamonts, macrogamonts and oocysts were located in large (up to 300 µm diameter) parasitophorous vacuoles of enterocytes in the ileum. Schizonts were not found. Review of previous reports suggests that multinucleated microgamonts have been mistaken for schizonts. Gametogonic development described in the present study can serve as a guide for differential diagnosis of Eimeria species in the histological sections of intestines.

High-Content Imaging Reveals Expansion of the Endosomal Compartment during Coxiella burnetii Parasitophorous Vacuole Maturation.

Coxiella burnetii is an obligate intracellular pathogen and the causative agent of human Q fever. Replication of the bacterium within a large parasitophorous vacuole (PV) resembling a host phagolysosome is required for pathogenesis. PV biogenesis is a pathogen driven process that requires engagement of several host cell vesicular trafficking pathways to acquire vacuole components. The goal of this study was to determine if infection by C. burnetii modulates endolysosomal flux to potentially benefit PV formation. HeLa cells, infected with C. burnetii or left uninfected, were incubated with fluorescent transferrin (Tf) for 0–30 min, and the amount of Tf internalized by cells quantitated by high-content imaging. At 3 and 5 days, but not 1 day post-infection, the maximal amounts of fluorescent Tf internalized by infected cells were significantly greater than uninfected cells. The rates of Tf uptake and recycling were the same for infected and uninfected cells; however, residual Tf persisted in EEA.1 positive compartments adjacent to large PV after 30 min of recycling in the absence of labeled Tf. On average, C. burnetii-infected cells contained significantly more CD63-positive endosomes than uninfected cells. In contrast, cells containing large vacuoles generated by Chlamydia trachomatis exhibited increased rates of Tf internalization without increased CD63 expression. Our results suggest that C. burnetii infection expands the endosomal system to increase capacity for endocytic material. Furthermore, this study demonstrates the power of high-content imaging for measurement of cellular responses to infection by intracellular pathogens.

Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation.

Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. In this work we developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, we screened a collection of RNAi-expressing flies for anti-Leishmania defense factors. Notably, we found three CD36-like scavenger receptors that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection.

Cysteine Peptidase B Regulates Leishmania mexicana Virulence through the Modulation of GP63 Expression.

Cysteine peptidases play a central role in the biology of Leishmania. In this work, we sought to further elucidate the mechanism(s) by which the cysteine peptidase CPB contributes to L. mexicana virulence and whether CPB participates in the formation of large communal parasitophorous vacuoles induced by these parasites. We initially examined the impact of L. mexicana infection on the trafficking of VAMP3 and VAMP8, two endocytic SNARE proteins associated with phagolysosome biogenesis and function. Using a CPB-deficient mutant, we found that both VAMP3 and VAMP8 were down-modulated in a CPB-dependent manner. We also discovered that expression of the virulence-associated GPI-anchored metalloprotease GP63 was inhibited in the absence of CPB. Expression of GP63 in the CPB-deficient mutant was sufficient to down-modulate VAMP3 and VAMP8. Similarly, episomal expression of GP63 enabled the CPB-deficient mutant to establish infection in macrophages, induce the formation of large communal parasitophorous vacuoles, and cause lesions in mice. These findings implicate CPB in the regulation of GP63 expression and provide evidence that both GP63 and CPB are key virulence factors in L. mexicana.

Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates.

BackgroundBovine besnoitiosis, caused by the protozoan Besnoitia besnoiti, reduces productivity and fertility of affected herds. Besnoitiosis continues to expand in Europe and no effective control tools are currently available. Experimental models are urgently needed. Herein, we describe for the first time the kinetics of standardised in vitro models for the B. besnoiti lytic cycle. This will aid to study the pathogenesis of the disease, in the screening for vaccine targets and drugs potentially useful for the treatment of besnoitiosis.MethodsWe compared invasion and proliferation of one B. tarandi (from Finland) and seven B. besnoiti isolates (Bb-Spain1, Bb-Spain2, Bb-Israel, Bb-Evora03, Bb-Ger1, Bb-France, Bb-Italy2) in MARC-145 cell culture. Host cell invasion was studied at 4, 6, 8 and 24 h post infection (hpi), and proliferation characteristics were compared at 24, 48, 72, 96, 120, and 144 hpi.ResultsIn Besnoitia spp., the key parameters that determine the sequential adhesion-invasion, proliferation and egress steps are clearly distinct from those in the related apicomplexans Toxoplasma gondii and Neospora caninum. Besnoitia spp. host cell invasion is a rather slow process, since only 50 % of parasites were found intracellular after 3–6 h of exposure to host cells, and invasion still took place after 24 h. Invasion efficacy was significantly higher for Bb-France, Bb-Evora03 and Bb-Israel. In addition, the time span for endodyogeny to take place was as long as 18–35 h. Bb-Israel and B. tarandi isolates were most prolific, as determined by the tachyzoite yield at 72 hpi. The total tachyzoite yield could not be predicted neither by invasion-related parameters (velocity and half time invasion) nor by proliferation parameters (lag phase and doubling time (dT)). The lytic cycle of Besnoitia was asynchronous as evidenced by the presence of three different plaque-forming tachyzoite categories (lysis plaques, large and small parasitophorous vacuoles).ConclusionsThis study provides first insights into the lytic cycle of B. besnoiti isolates and a standardised in vitro model that allows screening of drug candidates for the treatment of besnoitiosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1405-9) contains supplementary material, which is available to authorized users.

Development of an Ex Vivo Tissue Platform To Study the Human Lung Response to Coxiella burnetii.

Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute debilitating flu-like illness that can also present as chronic endocarditis. Disease typically occurs following inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In human cells, C. burnetii generates a replication niche termed the parasitophorous vacuole (PV) by directing fusion with autophagosomes and lysosomes. C. burnetii requires this lysosomal environment for replication and uses a Dot/Icm type IV secretion system to generate the large PV. However, we do not understand how C. burnetii evades the intracellular immune surveillance that triggers an inflammatory response. We recently characterized human alveolar macrophage (hAM) infection in vitro and found that avirulent C. burnetii triggers sustained interleukin-1β (IL-1β) production. Here, we evaluated infection of ex vivo human lung tissue, defining a valuable approach for characterizing C. burnetii interactions with a human host. Within whole lung tissue, C. burnetii preferentially replicated in hAMs. Additionally, IL-1β production correlated with formation of an apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)-dependent inflammasome in response to infection. We also assessed potential activation of a human-specific noncanonical inflammasome and found that caspase-4 and caspase-5 are processed during infection. Interestingly, although inflammasome activation is closely linked to pyroptosis, lytic cell death did not occur following C. burnetii-triggered inflammasome activation, indicating an atypical response after intracellular detection. Together, these studies provide a novel platform for studying the human innate immune response to C. burnetii.

The Early Secretory Pathway Contributes to the Growth of the Coxiella -Replicative Niche

Coxiella burnetii is a Gram-negative obligate intracellular bacterium. After internalization, this bacterium replicates in a large parasitophorous vacuole that has features of both phagolysosomes and autophagosomal compartments. We have previously demonstrated that early after internalization Coxiella phagosomes interact with both the endocytic and the autophagic pathways. In this report, we present evidence that the Coxiella-replicative vacuoles (CRVs) also interact with the secretory pathway. Rab1b is a small GTPase responsible for the anterograde transport between the endoplasmic reticulum and the Golgi apparatus. We present evidence that Rab1b is recruited to the CRV at later infection times (i.e., after 6 h of infection). Interestingly, knockdown of Rab1b altered vacuole growth, indicating that this protein was required for the proper biogenesis of the CRV. In addition, overexpression of the active GTPase-defective mutant (GFP-Rab1b Q67L) affected the development of the Coxiella-replicative compartment inhibiting bacterial growth. On the other hand, disruption of the secretory pathway by brefeldin A treatment or by overexpression of Sar1 T39N, a defective dominant-negative mutant of Sar1, affected the typical spaciousness of the CRVs. Taken together, our results show for the first time that the Coxiella-replicative niche also intercepts the early secretory pathway.

Actin Dynamics and Rho GTPases Regulate the Size and Formation of Parasitophorous Vacuoles Containing Coxiella burnetii

Q fever is a disease caused by Coxiella burnetii. In the host cell, this pathogen generates a large parasitophorous vacuole (PV) with lysosomal characteristics. Here we show that F-actin not only is recruited to but also is involved in the formation of the typical PV. Treatment of infected cells with F-actin-depolymerizing agents alters PV development. The small PVs formed in latrunculin B-treated cells were loaded with transferrin and Lysotracker and labeled with an antibody against cathepsin D, suggesting that latrunculin B did not affect vacuole cargo and its lysosomal characteristics. Nevertheless, the vacuoles were unable to fuse with latex bead phagosomes. It is known that actin dynamics are regulated by the Rho family GTPases. To assess the role of these GTPases in PV formation, infected cells were transfected with pEGFP expressing wild-type and mutant Rac1, Cdc42, and RhoA proteins. Rac1 did not show significant PV association. In contrast, PVs were decorated by both the wild types and constitutively active mutants of Cdc42 and RhoA. This association was inhibited by treatment of infected cells with chloramphenicol, suggesting a role for bacterial protein synthesis in the recruitment of these proteins. Interestingly, a decrease in vacuole size was observed in cells expressing dominant-negative RhoA; however, these small vacuoles accumulated transferrin, Lysotracker, and DQ-BSA. In summary, these results suggest that actin, likely modulated by the GTPases RhoA and Cdc42 and by bacterial proteins, is involved in the formation of the typical PV.

Microbicidal property of B1 cell derived mononuclear phagocyte

A mononuclear phagocyte derived from B1b cells (B1CDP) has been described. As these cells migrate from the peritoneal cavity to non-specific inflammatory lesion sites and are highly phagocytic via Fc and mannose receptors, their microbicidal ability of these cells was investigated using the Coxiella burnetii cell infection model in vitro. In this report, the pattern of infection and C. burnetii phase II survival in B1CDP phagosomes was compared with the pattern of infection of peritoneal macrophages from Xid mice (PMφ) and bone marrow derived macrophages (BMMφ). Infection was assessed by determining the large parasitophorous vacuole formation, the relative focus forming units and the quantification of DAPI (4′,6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy. When compared to macrophages, B1CDP are more permissive to the bacterial infection and less effective to kill them. Further, results suggest that IL-10 secreted by B1 cells are involved in their susceptibility to infection by C. burnetti, since B1CDP from IL-10 KO mice are more competent to control C. burnetii infection than cells from wild type mice. These data contribute further to characterize B1CDP as a novel mononuclear phagocyte.

Control of Parasitophorous Vacuole Expansion by LYST/Beige Restricts the Intracellular Growth of Leishmania amazonensis

The intracellular protozoan Leishmania replicates in parasitophorous vacuoles (PV) that share many features with late endosomes/lysosomes. L. amazonensis PVs expand markedly during infections, but the impact of PV size on parasite intracellular survival is still unknown. Here we show that host cells infected with L. amazonensis upregulate transcription of LYST/Beige, which was previously shown to regulate lysosome size. Mutations in LYST/Beige caused further PV expansion and enhanced L. amazonensis replication. In contrast, LYST/Beige overexpression led to small PVs that did not sustain parasite growth. Treatment of LYST/Beige over-expressing cells with vacuolin-1 reversed this phenotype, expanding PVs and promoting parasite growth. The opposite was seen with E-64d, which reduced PV size in LYST-Beige mutant cells and inhibited L. amazonensis replication. Enlarged PVs appear to protect parasites from oxidative damage, since inhibition of nitric oxide synthase had no effect on L. amazonensis viability within large PVs, but enhanced their growth within LYST/Beige-induced small PVs. Thus, the upregulation of LYST/Beige in infected cells functions as a host innate response to limit parasite growth, by reducing PV volume and inhibiting intracellular survival.

Efficient Method of Cloning the Obligate Intracellular Bacterium Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterium that replicates in a large lysosome-like parasitophorous vacuole (PV). Current methods of cloning C. burnetii are laborious and technically demanding. We have developed an alternative cloning method that involves excision of individual C. burnetii-laden PVs from infected cell monolayers by micromanipulation. To demonstrate the cloning utility and efficiency of this procedure, we coinfected Vero cells with isogenic variants of the Nine Mile strain of C. burnetii. Coinhabited PVs harboring Nine Mile phase II (NMII) and Nine Mile phase I (NMI) or Nine Mile crazy (NMC) were demonstrated by immunofluorescence. PVs were then randomly excised from cells coinfected with NMI and NMC by micromanipulation, and PVs harboring both strains were identified by PCR. Fresh Vero cells were subsequently infected with organisms from coinhabited PVs, and the PV excision and PCR screening process was repeated. Without exception, PVs obtained from second-round excisions contained clonal populations of either NMII or NMC, demonstrating that micromanipulation is an efficient and reproducible procedure for obtaining C. burnetii clones.

Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetii

Most intracellular parasites employ sophisticated mechanisms to direct biogenesis of a vacuolar replicative niche that circumvents default maturation through the endolysosomal cascade. However, this is not the case of the Q fever bacterium, Coxiella burnetii. This hardy, obligate intracellular pathogen has evolved to not only survive, but to thrive, in the harshest of intracellular compartments: the phagolysosome. Following internalization, the nascent Coxiella phagosome ultimately develops into a large and spacious parasitophorous vacuole (PV) that acquires lysosomal characteristics such as acidic pH, acid hydrolases and cationic peptides, defences designed to rid the host of intruders. However, transit of Coxiella to this environment is initially stalled, a process that is apparently modulated by interactions with the autophagic pathway. Coxiella actively participates in biogenesis of its PV by synthesizing proteins that mediate phagosome stalling, autophagic interactions, and development and maintenance of the mature vacuole. Among the potential mechanisms mediating these processes is deployment of a type IV secretion system to deliver effector proteins to the host cytosol. Here we summarize our current understanding of the cellular events that occur during parasitism of host cells by Coxiella.

Replication of Coxiella burnetii Is Inhibited in CHO K‐1 Cells Treated with Inhibitors of Cholesterol Metabolism

Coxiella burnetii directs the synthesis of a large parasitophorous vacuole (PV) that is required for its replication. While some lysosomal characteristics of the PV have been described, the origin and composition of the PV membrane remain largely undefined. Cholesterol is an essential component of mammalian membranes where it lends mechanical stability and serves as a platform for signaling proteins. Using infected Chinese hamster ovary cells as a model, we examined whether cholesterol is trafficked to the C. burnetii PV membrane and the effects of inhibitors of cholesterol metabolism on C. burnetii replication. When infected cells were stained with filipin, a fluorescent polyene antifungal agent that binds cholesterol, obvious staining of PV was observed indicating the PV membrane is cholesterol-rich. Furthermore, replication of C. burnetii was significantly inhibited in cells treated with the cholesterol metabolism inhibitors lovastatin, ketoconazole, imipramine, progesterone, and U18666A. These data suggest that cholesterol is an important component of the C. burnetii PV membrane and that normal cellular cholesterol metabolism is required for optimal C. burnetii replication.

Coxiella burnetii inhabits a cholesterol-rich vacuole and influences cellular cholesterol metabolism

Coxiella burnetii directs the synthesis of a large parasitophorous vacuole (PV) required for replication. While some lysosomal characteristics of the PV have been described, the origin and composition of the PV membrane is largely undefined. Cholesterol is an essential component of mammalian cell membranes where it plays important regulatory and structural roles. Here we investigated the role of host cholesterol in biogenesis and maintenance of the C. burnetii PV in Vero cells. The C. burnetii PV membrane stained with filipin and was positive for the lipid raft protein flotillin-1, suggesting PV membranes are enriched in cholesterol and contain lipid raft microdomains. C. burnetii infection increased host cell cholesterol content by 1.75-fold with a coincident upregulation of host genes involved in cholesterol metabolism. Treatment with U18666A, lovastatin, or 25-hydroxycholesterol, pharmacological agents that inhibit cholesterol uptake and/or biosynthesis, altered PV morphology and partially inhibited C. burnetii replication. Complete inhibition of C. burnetii PV development and replication was observed when infected cells were treated with imipramine or ketoconazole, inhibitors of cholesterol uptake and biosynthesis respectively. We conclude that C. burnetii infection perturbs host cell cholesterol metabolism and that free access to host cholesterol stores is required for optimal C. burnetii replication.

Maturation of the Coxiella burnetii parasitophorous vacuole requires bacterial protein synthesis but not replication.

This study examined whether protein synthesis and replication are required for maturation and fusogenicity of the lysosomal-like, large and spacious parasitophorous vacuole (PV) of Coxiella burnetii, an obligate intracellular bacterium. Large and spacious PV with multiple non-replicating C. burnetii were observed by phase microscopy in Vero cells infected at a multiplicity of infection of ten and treated with a bacteriostatic concentration of nalidixic acid or carbenicillin, antimicrobics that inhibit DNA and cell wall biosynthesis respectively. Conversely, large and spacious PV were not observed in cells treated with a bacteriostatic concentration of the protein synthesis inhibitor chloramphenicol. Rather, fluorescence microscopy of individual cells revealed multiple, acidic PV harbouring a single organism tightly bounded by a LAMP-1 positive vacuolar membrane. These vacuoles homotypically fused to form a large and spacious PV upon removal of the drug. Chloramphenicol also inhibited trafficking of latex beads to large and spacious PV and caused mature PV to collapse. Collectively, these results demonstrate that C. burnetii protein synthesis, but not replication, is required for fusion between nascent C. burnetii PV and latex bead phagosomes, and also for formation and maintenance of large and spacious, replicative PV. However, transit of nascent PV through the endocytic pathway to ultimately acquire lysosomal markers appears to occur irrespective of Coxiella protein synthesis.