Intracellular Life Research Articles

Norovirus gene expression and replication

Noroviruses are small, positive-sense RNA viruses within the family Caliciviridae, and are now accepted widely as a major cause of acute gastroenteritis in both developed and developing countries. Despite their impact, our understanding of the life cycle of noroviruses has lagged behind that of other RNA viruses due to the inability to culture human noroviruses (HuNVs). Our knowledge of norovirus biology has improved significantly over the past decade as a result of numerous technological advances. The use of a HuNV replicon, improved biochemical and cell-based assays, combined with the discovery of a murine norovirus capable of replication in cell culture, has improved greatly our understanding of the molecular mechanisms of norovirus genome translation and replication, as well as the interaction with host cell processes. In this review, the current state of knowledge of the intracellular life of noroviruses is discussed with particular emphasis on the mechanisms of viral gene expression and viral genome replication.

Systemic Spread and Propagation of a Plant-Pathogenic Virus in European Honeybees, Apis mellifera

ABSTRACTEmerging and reemerging diseases that result from pathogen host shifts are a threat to the health of humans and their domesticates. RNA viruses have extremely high mutation rates and thus represent a significant source of these infectious diseases. In the present study, we showed that a plant-pathogenic RNA virus, tobacco ringspot virus (TRSV), could replicate and produce virions in honeybees, Apis mellifera, resulting in infections that were found throughout the entire body. Additionally, we showed that TRSV-infected individuals were continually present in some monitored colonies. While intracellular life cycle, species-level genetic variation, and pathogenesis of the virus in honeybee hosts remain to be determined, the increasing prevalence of TRSV in conjunction with other bee viruses from spring toward winter in infected colonies was associated with gradual decline of host populations and winter colony collapse, suggesting the negative impact of the virus on colony survival. Furthermore, we showed that TRSV was also found in ectoparasitic Varroa mites that feed on bee hemolymph, but in those instances the virus was restricted to the gastric cecum of Varroa mites, suggesting that Varroa mites may facilitate the spread of TRSV in bees but do not experience systemic invasion. Finally, our phylogenetic analysis revealed that TRSV isolates from bees, bee pollen, and Varroa mites clustered together, forming a monophyletic clade. The tree topology indicated that the TRSVs from arthropod hosts shared a common ancestor with those from plant hosts and subsequently evolved as a distinct lineage after transkingdom host alteration. This study represents a unique example of viruses with host ranges spanning both the plant and animal kingdoms.

Expression of Porcine Respiratory and Reproductive Syndrome Virus Membrane-Associated Proteins in Listeria ivanovii via a Genome Site-Specific Integration and Expression System

Listeria ivanovii (LI) possesses a facultative intracellular life cycle in macrophages and non-professional phagocytes thus making it a potential novel vaccine vector. We have developed a genome site-specific integration system as a universal tool for constructing the live LI-based vaccines. Using this system, the porcine reproductive and respiratory syndrome virus (PRRSV) orf5 and orf6 genes, coding for PRRSV membrane-associated protein GP5 and M, respectively, were integrated into the LI genome. Genome PCR and sequencing results showed that the orf5 or orf6 gene had been integrated into the LI genome downstream of orfXYZ or mpl gene. Western blot analysis of the non-secreted and secreted bacterial protein samples showed the expression and secretion of GP5 or M protein from the recombinant LI strains. The secretion of the heterologous proteins is a key step for bacterial vaccine vectors to deliver the antigens to the immune system to stimulate antigen-specific immune responses. Therefore, this integration system may be a potential tool for constructing novel vaccines using live LI as vector.

An historical perspective on how advances in microscopic imaging contributed to understanding the Leishmania Spp. and Trypanosoma cruzi host-parasite relationship.

The literature has identified complex aspects of intracellular host-parasite relationships, which require systematic, nonreductionist approaches and spatial/temporal information. Increasing and integrating temporal and spatial dimensions in host cell imaging have contributed to elucidating several conceptual gaps in the biology of intracellular parasites. To access and investigate complex and emergent dynamic events, it is mandatory to follow them in the context of living cells and organs, constructing scientific images with integrated high quality spatiotemporal data. This review discusses examples of how advances in microscopy have challenged established conceptual models of the intracellular life cycles of Leishmania spp. and Trypanosoma cruzi protozoan parasites.

The phtC-phtD locus equips Legionella pneumophila for thymidine salvage and replication in macrophages.

The phagosomal transporter (Pht) family of the major facilitator superfamily (MFS) is encoded by phylogenetically related intracellular gammaproteobacteria, including the opportunistic pathogen Legionella pneumophila. The location of the pht genes between the putative thymidine kinase (tdk) and phosphopentomutase (deoB) genes suggested that the phtC and phtD loci contribute to thymidine salvage in L. pneumophila. Indeed, a phtC(+) allele in trans restored pyrimidine uptake to an Escherichia coli mutant that lacked all known nucleoside transporters, whereas a phtD(+) allele did not. The results of phenotypic analyses of L. pneumophila strains lacking phtC or phtD strongly indicate that L. pneumophila requires PhtC and PhtD function under conditions where sustained dTMP synthesis is compromised. First, in broth cultures that mimicked thymidine limitation or starvation, L. pneumophila exhibited a marked requirement for PhtC function. Conversely, mutation of phtD conferred a survival advantage. Second, in medium that lacked thymidine, multicopy phtC(+) or phtD(+) alleles enhanced the survival of L. pneumophila thymidylate synthase (thyA)-deficient strains, which cannot synthesize dTMP endogenously. Third, under conditions in which transport of the pyrimidine nucleoside analog 5-fluorodeoxyuridine (FUdR) would inhibit growth, PhtC and PhtD conferred a growth advantage to L. pneumophila thyA(+) strains. Finally, when cultured in macrophages, L. pneumophila required the phtC-phtD locus to replicate. Accordingly, we propose that PhtC and PhtD contribute to protect L. pneumophila from dTMP starvation during its intracellular life cycle.

Localization of H.pylori within the vacuole of Candida yeast by direct immunofluorescence technique.

Reports indicate that H.pylori is able to invade the eukaryotic cells and establish inside their vacuoles. In this study, FITC-conjugated IgY-Hp was used to localize H.pylori inside the vacuole of Candida yeast. Presence of intracellular H.pylori inside the new generations of yeast cells was also examined by light microscopy and Live/Dead BacLight staining method. A single colony of fresh yeast culture was cultivated in a 100-µl medium containing yeast extract and N-acetylglucoseamine supplemented with fetal bovine serum. After 12-hr incubation at 37℃, FITC-conjugated IgY-Hp was added. After 3 hours, 10 µL of yeast suspension was smeared on a glass slide, air-dried and examined by fluorescent microscopy. Wet mounts of yeast culture and Live/Dead BacLight stained preparations were examined by light and fluorescent microscopy, respectively. Photographs were taken from the fast-moving H.pylori inside the yeast vacuoles. Fluorescent microscopy showed that FITC-conjugated IgY-Hp could enter yeast cells and specifically react with H.pylori, localizing the bacterium inside the yeast vacuole. Photographs taken from wet mounts observed by light and fluorescent microscopy showed fast-moving H.pylori cells in the vacuole of mother as well as daughter yeast cells. The intravacuolar H.pylori cells stained green, showing their viability. Intracellular life of prokaryotes inside eukaryotes has been described as an evolutionary phenomenon with a great impact on bacterial persistence despite environmental stresses. Results of this study demonstrated the specific interaction of FITC-conjugated IgY-Hp with H.pylori cells and the bacterial localization inside the Candida yeast vacuole. The intracellular bacteria were viable and existed in the vacuole of next generations of yeast cells. It appears that H.pylori is well-equipped to dwell within the vacuole of eukaryotic cells where it is protected from stressful conditions, including antibacterial therapy. Presence of H.pylori inside the vacuole of new generations of yeasts demonstrates the intimate relationship between the two microorganisms, resulting in bacterial inheritance as part of the vacuolar content of yeast cells.

Burkholderia cenocepacia J2315 escapes to the cytosol and actively subverts autophagy in human macrophages

Selective autophagy functions to specifically degrade cellular cargo tagged by ubiquitination, including bacteria. Strains of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that cause life-threatening infections in patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD). While there is evidence that defective macrophage autophagy in a mouse model of CF can influence B. cenocepacia susceptibility, there have been no comprehensive studies on how this bacterium is sensed and targeted by the host autophagy response in human macrophages. Here, we describe the intracellular life cycle of B. cenocepacia J2315 and its interaction with the autophagy pathway in human cells. Electron and confocal microscopy analyses demonstrate that the invading bacteria interact transiently with the endocytic pathway before escaping to the cytosol. This escape triggers theselective autophagy pathway, and the recruitment of ubiquitin, the ubiquitin-binding adaptors p62 and NDP52 and the autophagosome membrane-associated protein LC3B, to the bacterial vicinity. However, despite recruitment of these key autophagy pathway effectors, B. cenocepacia blocks autophagosome completion and replicates in the host cytosol. We find that a pre-infection increase in cellular autophagy flux can significantly inhibit B. cenocepacia replication and that lower autophagy flux in macrophages from immunocompromised CGD patients could contribute to increased B. cenocepacia susceptibility, identifying autophagy manipulation as a potential therapeutic approach to reduce bacterial burden in B. cenocepacia infections.

Identification of a type IV secretion substrate ofBrucella abortusthat participates in the early stages of intracellular survival

Brucella abortus, the aetiological agent of bovine brucellosis, is an intracellular pathogen whose virulence is completely dependent on a type IV secretion system. This secretion system translocates effector proteins into the host cell to modulate the intracellular fate of the bacterium in order to establish a secure niche were it actively replicates. Although much has been done in understanding how this secretion system participates in the virulence process, few effector proteins have been identified to date. We describe here the identification of a type IV secretion substrate (SepA) that is only present in Brucella spp. and has no detectable homology to known proteins. This protein is secreted in a virB-dependent manner in a two-step process involving a periplasmic intermediate and secretion is necessary for its function. The deletion mutant showed a defect in the early stages of intracellular replication in professional and non-professional phagocytes although it invades the cells more efficiently than the wild-type parental strain. Our results indicate that, even though the mutant was more invasive, it had a defect in excluding the lysosomal marker Lamp-1 and was inactivated more efficiently during the early phases of the intracellular life cycle.

In vitro and in vivo models to study human listeriosis: mind the gap

Listeria monocytogenes (Lm) is the etiological agent of listeriosis, one of the deadliest human foodborne infections. Lm is able to cross the intestinal, placental and blood–brain barriers, leading to septicemia, fetoplacental infection, meningitis and encephalitis. The intracellular life cycle of this facultative intracellular bacterium has been studied in detail in in vitro cell culture systems. Lm enters in non-phagocytic cells in an InlA–Ecad and/or InB-Met dependent manner. Lm then escapes from its internalization vacuole via the action of the pore forming toxin LLO and spreads from cell to cell in an ActA-dependent manner. In vivo studies in “humanized” mouse models and human epidemiological data have led to the deciphering of the mechanisms underlying Lm crossing of intestinal and placental barriers. As illustrated in this review, in vivo outcomes could not be deduced directly from in vitro observations, and led to revisit the intracellular fate of Lm during infection.

The emergence of drug resistant HIV variants at virological failure of HAART combinations containing efavirenz, tenofovir and lamivudine or emtricitabine within the UK Collaborative HIV Cohort

Lamivudine (3TC) and emtricitabine (FTC) are guideline choices for combination highly active antiretroviral therapy (HAART). 3TC has a shorter intracellular half life than FTC and may be more likely to lead to the development of drug resistant HIV variants. In this study we analysed linked data from the observational UK Collaborative HIV Cohort (CHIC) Study and UK HIV Drug Resistance Database (HDRD) to investigate the rate of development of K65R or M184V resistance mutations in patients failing on combinations containing tenofovir (TDF) and efavirenz (EFV) with either 3TC or FTC. Virological failure was defined as 1 viral load >400copies/ml. Rates were stratified by demographic variables, baseline viral load, current CD4 count, current viral load and year of starting regimen. Significant associations were identified using Poisson regression models and multivariable analyses were performed adjusting for the variables above. Logistic regression was used to determine whether there were any significant associations between type of regimen and detection of resistance mutation. 5455 patients received either (or both) 3TC, TDF and EFV or FTC, TDF and EFV contributing 6465 treatment episodes over 9962 person-years follow up. 47 of these episodes were preceded by resistance tests showing development of K65R or M184V mutation and were hence excluded. The majority of treatment episodes consisted of FTC- (n=5190) rather than 3TC- (n=1228) based regimens. 21 cases of K65R were detected over the course of follow up, giving an overall event rate of 0.21 (95% CI: 0.12-0.31)/100 person years follow up (PYFU). The overall event rate for detection of M184V was 0.38 (95% CI: 0.26-0.5)/100 PYFU. 201 patients receiving either regimen for the first time experienced virological failure. Of those receiving 3TC (n=53), 7 (13.2%), 12 (22.6%) and 15 (28.3%) developed K65R, M184V and either K65R or M184V respectively. Of those receiving FTC (n=148), 13 (8.8%), 20 (13.5%) and 26 (17.6%) developed K65R, M184V and either K65R or M184V respectively. Although patients on 3TC were more likely to develop resistance, this was not statistically significant in univariable (OR 1.85 (95% CI: 0.89-3.85, p=0.09)) or multivariable analyses (OR 1.89 (95% CI: 0.89-4.01, p=0.1)). We have not found evidence of an increased risk of development of M184V and K65R in patients exposed to 3TC.

The Intracellular Life ofCryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen with worldwide distribution. Serological studies of human populations show a high prevalence of human infection, which rarely progresses to disease in immunocompetent hosts. However, decreased host immunity places individuals at high risk for cryptococcal disease. The disease can result from acute infection or reactivation of latent infection, in which yeasts within granulomas and host macrophages emerge to cause disease. In this review, we summarize what is known about the cellular recognition, ingestion, and killing of C. neoformans and discuss the unique and remarkable features of its intracellular life, including the proposed mechanisms for fungal persistence and killing in phagocytic cells.

Chlamydia bacteriophages

Phages are called "good viruses" due to their ability to infect and kill pathogenic bacteria. Chlamydia are small, Gram-negative (G-) microbes that can be dangerous to human and animals. In humans, these bacteria are etiological agents of diseases such as psittacosis or respiratory tract diseases, while in animals, the infection may result in enteritis in cattle and chronic bowel diseases, as well as miscarriages in sheep. The first-known representative of chlamydiaphages was Chp1. It was discovered in Chlamydia psittaci isolates. Since then, four more species of chlamydiaphages have been identified [Chp2, Chp3, φCPG1 φCPAR39 (φCpn1) and Chp4]. All of them were shown to infect Chlamydia species. This paper described all known chlamydiaphages. They were characterised in terms of origin, host range, and their molecular structure. The review concerns the characterisation of bacteriophages that infects pathogenic and dangerous bacteria with unusual, intracellular life cycles that are pathogenic. In the era of antibiotic resistance, it is difficult to cure chlamydophilosis. Those bacteriophages can be an alternative to antibiotics, but before this happens, we need to get to know chlamydiaphages better.

Hidden Behind Autophagy: The Unconventional Roles of ATG Proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular catabolic transport route that generally allows the lysosomal degradation of cytoplasmic components, including bulk cytosol, protein aggregates, damaged or superfluous organelles and invading microbes. Target structures are sequestered by double‐membrane vesicles called autophagosomes, which are formed through the concerted action of the autophagy (ATG)‐related proteins. Until recently it was assumed that ATG proteins were exclusively involved in autophagy. A growing number of studies, however, have attributed functions to some of them that are distinct from their classical role in autophagosome biogenesis. Autophagy‐independent roles of the ATG proteins include the maintenance of cellular homeostasis and resistance to pathogens. For example, they assist and enhance the turnover of dead cells and microbes upon their phagocytic engulfment, and inhibit murine norovirus replication. Moreover, bone resorption by osteoclasts, innate immune regulation triggered by cytoplasmic DNA and the ER‐associated degradation regulation all have in common the requirement of a subset of ATG proteins. Microorganisms such as coronaviruses, Chlamydia trachomatis or Brucella abortus have even evolved ways to manipulate autophagy‐independent functions of ATG proteins in order to ensure the completion of their intracellular life cycle. Taken together these novel mechanisms add to the repertoire of functions and extend the number of cellular processes involving the ATG proteins.

Living cell as a universal computer

A number of similarities between living cells and universal computers are considered. Intracellular life processes are compared to computation processes inside a computer. Proteins coordinating intracellular processes are associated with computer programs during runtime. The possibility of the existence of a genetic programming language that allows effectively building new genes from the existing ones is considered.

Rickettsia typhi Possesses Phospholipase A2 Enzymes that Are Involved in Infection of Host Cells

The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA2 activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.

Involvement of lipid rafts in the budding-like exit of Orientia tsutsugamushi

Orientia tsutsugamushi is an intracellular parasite that causes scrub typhus. After entering the cytoplasm by induced phagocytosis, O. tsutsugamushi escapes from the primary phagosome into the host cytosol, where it replicates slowly. Subsequently, it is released from the host cells by a process resembling viral budding with a remaining bacterial aggregate near the nucleus. Lipid rafts have been implicated in the life cycle of a wide variety of pathogenic microorganisms. We have observed that proteins of O. tsutsugamushi were co-fractionated with the lipid rafts over a sucrose density gradient, suggesting the possible involvement of lipid rafts during the intracellular life cycle of O. tsutsugamushi. The entry of O. tsutsugamushi into the host cells was shown to be independent on lipid rafts as judged by the inability of lipid raft-disrupting agents to inhibit bacterial entry and no co-localization of bacterial proteins with caveolin. To our interest, a 47-kDa protein (HtrA) was observed to be co-localized with caveolin at the cell membrane at 72 h after infection, when bacterial particles move to the cell membrane and initiate the exit into the extracellular environment. Our results suggest that O. tsutsugamushi involves lipid rafts of the host cells in the budding-like process to exit from host cells.

Key Role for the Alternative Sigma Factor, SigH, in the Intracellular Life of Mycobacterium avium subsp. paratuberculosis during Macrophage Stress

Mycobacterium avium subsp. paratuberculosis causes Johne's disease, an enteric infection in cattle and other ruminants, greatly afflicting the dairy industry worldwide. Once inside the cell, M. avium subsp. paratuberculosis is known to survive harsh microenvironments, especially those inside activated macrophages. To improve our understanding of M. avium subsp. paratuberculosis pathogenesis, we examined phagosome maturation associated with transcriptional responses of M. avium subsp. paratuberculosis during macrophage infection. Monitoring cellular markers, only live M. avium subsp. paratuberculosis bacilli were able to prevent phagosome maturation and reduce its acidification. On the transcriptional level, over 300 M. avium subsp. paratuberculosis genes were significantly and differentially regulated in both naive and IFN-γ-activated macrophages. These genes include the sigma factor H (sigH) that was shown to be important for M. avium subsp. paratuberculosis survival inside gamma interferon (IFN-γ)-activated bovine macrophages. Interestingly, an sigH-knockout mutant showed increased sensitivity to a sustained level of thiol-specific oxidative stress. Large-scale RNA sequence analysis revealed that a large number of genes belong to the sigH regulon, especially following diamide stress. Genes involved in oxidative stress and virulence were among the induced genes in the sigH regulon with a putative consensus sequence for SigH binding that was recognized in a subset of these genes (n = 30), suggesting direct regulation by SigH. Finally, mice infections showed a significant attenuation of the ΔsigH mutant compared to its parental strain, suggesting a role for sigH in M. avium subsp. paratuberculosis virulence. Such analysis could identify potential targets for further testing as vaccine candidates against Johne's disease.

Mechanisms of Francisella tularensis Intracellular Pathogenesis

Francisella tularensis is a zoonotic intracellular pathogen and the causative agent of the debilitating febrile illness tularemia. Although natural infections by F. tularensis are sporadic and generally localized, the low infectious dose, with the ability to be transmitted to humans via multiple routes and the potential to cause life-threatening infections, has led to concerns that this bacterium could be used as an agent of bioterror and released intentionally into the environment. Recent studies of F. tularensis and other closely related Francisella species have greatly increased our understanding of mechanisms used by this organism to infect and cause disease within the host. Here, we review the intracellular life cycle of Francisella and highlight key genetic determinants and/or pathways that contribute to the survival and proliferation of this bacterium within host cells.

The Francisella tularensis LVS ΔpdpCmutant exhibits a unique phenotype during intracellular infection

BackgroundA prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system.ResultsWe characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1β, albeit lower than LVS, and markedly induced LDH release.ConclusionsThe phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.

Poxvirus membrane biogenesis: rupture not disruption

Enveloped viruses acquire their membrane from the host by budding at, or wrapping by, cellular membranes. Transmission electron microscopy (TEM) images, however, suggested that the prototype member of the poxviridae, vaccinia virus (VACV), may create its membrane 'de novo' with free open ends exposed in the cytosol. Within the frame of the German-wide priority programme we re-addressed the biogenesis and origin of the VACV membrane using electron tomography (ET), cryo-EM and lipid analysis of purified VACV using mass spectrometry (MS). This review discussed how our data led to a model of unconventional membrane biogenesis involving membrane rupture and the generation of a single open membrane from open membrane intermediates. Lipid analyses of purified virus by MS suggest an ER origin with a relatively low cholesterol content compared with whole cells, confirming published data. Unlike previous reports using thin-layer chromatography, no depletion of phosphatidylethanolamine was detected. We did detect, however, an enrichment for phosphatidic acid, diacylglycerol and phosphatidylinositol in the virion. Our data are discussed in the light of other pathogens that may requirecellular membrane rupture during their intracellular life cycle.