Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.

Living on the edge: parasite taxonomy in Australia

The way in which the huge Australian parasite fauna is described (discovery and naming) is the subject of this address. The approach to the task has never been well-organised so that a few groups of parasites are now relatively well-known because of the efforts of small groups of workers who have made sustained efforts in these groups, but equally some host-parasite systems have been almost completely ignored in that no worker has ever given them sustained attention. A high proportion of Australian parasites have been described by international workers; The sustaining of interest in a group of parasites over a long period is the key to real progress being made. The nature of the organisation of Australian science presently means that few positions are available for parasite taxonomists and funding for taxonomic research is scarce. Thus, parasite taxonomy (like the taxonomy of many groups of Australian plants and animals) can only be considered to be in crisis. (C) 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Host-bacteria interactions : Host cell responses and bacterial pathogenesis

Helicobacter pylori colonizes the human stomach, where it causes gastritis that may develop into peptic ulcer disease or cancer when left untreated. Neisseria gonorrhoeae colonizes the urogenital tract and causes the sexually transmitted disease gonorrhea. In contrast, Lactobacillus species are part of the human microbiota, which is the resident microbial community, and are considered to be beneficial for health. The first host cell types that bacteria encounter when they enter the body are epithelial cells, which form the border between the inside and the outside, and macrophages, which are immune cells that engulf unwanted material.       The focus of this thesis has been the interaction between the host and bacteria, aiming to increase our knowledge of the molecular mechanisms that underlie the host responses and their effects on bacterial pathogenicity. Understanding the interactions between bacteria and the host will hopefully enable the development of new strategies for the treatment of infectious disease. In paper I, we investigated the effect of N. gonorrhoeae on the growth factor amphiregulin in cervical epithelial cells and found that the processing and release of amphiregulin changes upon infection. In paper II, we examined the expression of the transcription factor early growth response-1 (EGR1) in epithelial cells during bacterial colonization. We demonstrated that EGR1 is rapidly upregulated by many different bacteria. This upregulation is independent of the pathogenicity, Gram-staining type and level of adherence of the bacteria, but generally requires viable bacteria and contact with the host cell. The induction of EGR1 is mediated primarily by signaling through EGFR, ERK1/2 and β1-integrins. In paper III, we described the interactions of the uncharacterized protein JHP0290, which is secreted by H. pylori, with host cells. JHP0290 is able to bind to several cell types and induces apoptosis and TNF release in macrophages. For both of these responses, signaling through Src family kinases and ERK is essential. Apoptosis is partially mediated by TNF release. Finally, in paper IV, we showed that certain Lactobacillus strains can reduce the colonization of H. pylori on gastric epithelial cells. Lactobacilli decrease the gene expression of SabA and thereby inhibit the binding mediated by this adhesin.

Regulation of Rab5 GTPase activity during Pseudomonas aeruginosa-macrophage interaction

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Several antibiotic resistant strains of P. aeruginosa are commonly found as secondary infection in immune-compromised patients leaving significant mortality and healthcare cost. Pseudomonas aeruginosa successfully avoids the process of phagocytosis, the first line of host defense, by secreting several toxic effectors. Effectors produced from P. aeruginosa Type III secretion system are critical molecules required to disrupt mammalian cell signaling and holds particular interest to the scientists studying host-pathogen interaction. Exoenzyme S (ExoS) is a bi-functional Type III effector that ADP-ribosylates several intracellular Ras (Rat sarcoma) and Rab (Response to abscisic acid) small GTPases in targeted host cells. The Rab5 protein acts as a rate limiting protein during phagocytosis by switching from a GDP- bound inactive form to a GTP-bound active form. Activation and inactivation of Rab5 protein is regulated by several Rab5-GAPs (GTPase Activating Proteins) and Rab5-GEFs (Rab5-Guanine nucleotide Exchange Factors). Some pathogenic bacteria have shown affinity for Rab proteins during infection and make their way inside the cell. This dissertation demonstrated that Rab5 plays a critical role during early steps of P. aeruginosa invasion in J774-Eclone macrophages. It was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis that occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and more than one arginine sites in Rab5 are possible targets for ADP-ribosylation modification. However, the expression of Rin1, but not other Rab5GEFs (Rabex-5 and Rap6) reversed this down-regulation of Rab5 in vivo. Further studies revealed that the C-terminus of Rin1 carrying Rin1:Vps9 and Rin1:RA domains are required for optimal Rab5 activation in conjunction with active Ras. These observations demonstrate a novel mechanism of Rab5 targeting to phagosome via Rin1 during the phagocytosis of P. aeruginosa. The second part of this dissertation investigated antimicrobial activities of Dehydroleucodine (DhL), a secondary metabolite from Artemisia douglasiana, against P. aeruginosa growth and virulence. Populations of several P. aeruginosa strains were completely susceptible to DhL at a concentration between 0.48~0.96 mg/ml and treatment at a threshold concentration (0.12 mg/ml) inhibited growth and many virulent activities without damaging the integrity of the cell suggesting anti-Pseudomonas activity of DhL.

Annotation of Gyrodactylus salaris transcriptome

Relationship between organisms within an ecosystem is one of the main focuses in the study of ecology and evolution. For instance, host-parasite interactions have long been under close interest of ecology, evolutionary biology and conservation science, due to great variety of strategies and interaction outcomes. The monogenean ecto-parasites consist of a significant portion of flatworms. Gyrodactylus salaris is a monogenean freshwater ecto-parasite of Atlantic salmon (Salmo salar) whose damage can make fish to be prone to further bacterial and fungal infections. G. salaris is the only one parasite whose genome has been studied so far. The RNA-seq data analyzed in this thesis has already been annotated by using LAST. The RNA-seq data was obtained from Illumina sequencing i.e. yielded reads were assembled into 15777 transcripts. Last resulted in annotation of 46% transcripts and remaining were left unknown. This thesis work was started with whole data and annotation process was continued by the use of PANNZER, CDD and InterProScan. This annotation resulted in 56% successfully annotated sequences having parasite specific proteins identified. This thesis represents the first of Monogenean transcriptomic information which gives an important source for further research on this specie. Additionally, comparison of annotation methods interestingly revealed that description and domain based methods perform better than simple similarity search methods. Therefore it is more likely to suggest the use of these tools and databases for functional annotation. These results also emphasize the need for use of multiple methods and databases. It also highlights the need of more genomic information related to G. salaris.

Comparative transcriptome profiling of human foreskin fibroblasts infected with the Sylvio and Y strains of Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas Disease, is phylogenetically distributed into nearly identical genetic strains which show divergent clinical presentations including differences in rates of cardiomyopathy in humans, different vector species and transmission cycles, and differential congenital transmission in a mouse model. The population structure of these strains divides into two groups, which are geographically and clinically distinct. The aim of this study was to compare the transcriptome of two strains of T. cruzi, Sylvio vs. Y to identify differences in expression that could account for clinical and biochemical differences. We collected and sequenced RNA from T. cruzi-infected and control Human Foreskin Fibroblasts at three timepoints. Differential expression analysis identified gene expression profiles at different timepoints in Sylvio infections, and between Sylvio and Y infections in both parasite and host. The Sylvio strain parasite and the host response to Sylvio infection largely mirrored the host-pathogen interaction seen in our previous Y strain work. IL-8 was more highly expressed in Sylvio-infected HFFs than in Y-infected HFFs.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

International audience

Migration, prospecting, dispersal? What host movement matters for infectious agent circulation?

Spatial disease ecology is emerging as a new field that requires the integration of complementary approaches to address how the distribution and movements of hosts and parasites may condition the dynamics of their interactions. In this context, migration, the seasonal movement of animals to different zones of their distribution, is assumed to play a key role in the broad scale circulation of parasites and pathogens. Nevertheless, migration is not the only type of host movement that can influence the spatial ecology, evolution, and epidemiology of infectious diseases. Dispersal, the movement of individuals between the location where they were born or bred to a location where they breed, has attracted attention as another important type of movement for the spatial dynamics of infectious diseases. Host dispersal has notably been identified as a key factor for the evolution of host-parasite interactions as it implies gene flow among local host populations and thus can alter patterns of coevolution with infectious agents across spatial scales. However, not all movements between host populations lead to dispersal per se. One type of host movement that has been neglected, but that may also play a role in parasite spread is prospecting, i.e., movements targeted at selecting and securing new habitat for future breeding. Prospecting movements, which have been studied in detail in certain social species, could result in the dispersal of infectious agents among different host populations without necessarily involving host dispersal. In this article, we outline how these various types of host movements might influence the circulation of infectious disease agents and discuss methodological approaches that could be used to assess their importance. We specifically focus on examples from work on colonial seabirds, ticks, and tick-borne infectious agents. These are convenient biological models because they are strongly spatially structured and involve relatively simple communities of interacting species. Overall, this review emphasizes that explicit consideration of the behavioral and population ecology of hosts and parasites is required to disentangle the relative roles of different types of movement for the spread of infectious diseases.

Evaluation of glucose dehydrogenase and pyrroloquinoline quinine (pqq) mutagenesis that renders functional inadequacies in host plants

The rhizospheric zone abutting plant roots usually clutches a wealth of microbes. In the recent past, enormous genetic resources have been excavated with potential applications in host plant interaction and ancillary aspects. Two Pseudomonas strains were isolated and identified through 16S rRNA and rpoD sequence analyses as P. fluorescens QAU67 and P. putida QAU90. Initial biochemical characterization and their root-colonizing traits indicated their potential role in plant growth promotion. Such aerobic systems, involved in gluconic acid production and phosphate solubilization, essentially require the pyrroloquinoline quinine (PQQ)- dependent glucose dehydrogenase (GDH) in the genome. The PCR screening and amplification of GDH and PQQ and subsequent induction of mutagenesis characterized their possible role as antioxidants as well as in growth promotion, as probed in vitro in lettuce and in vivo in rice, bean, and tomato plants. The results showed significant differences (p ≤ 0.05) in parameters of plant height, fresh weight, and dry weight, etc., deciphering a clear and in fact complementary role of GDH and PQQ in plant growth promotion. Our study not only provides direct evidence of the in vivo role of GDH and PQQ in host plants but also reveals their functional inadequacy in the event of mutation at either of these loci.

MS-based metabolomics facilitates the discovery of in vivo functional small molecules with a diversity of biological contexts

In vivo small molecules as necessary intermediates are involved in numerous critical metabolic pathways and biological processes associated with many essential biological functions and events. There is growing evidence that MS-based metabolomics is emerging as a powerful tool to facilitate the discovery of functional small molecules that can better our understanding of development, infection, nutrition, disease, toxicity, drug therapeutics, gene modifications and host-pathogen interaction from metabolic perspectives. However, further progress must still be made in MS-based metabolomics because of the shortcomings in the current technologies and knowledge. This technique-driven review aims to explore the discovery of in vivo functional small molecules facilitated by MS-based metabolomics and to highlight the analytic capabilities and promising applications of this discovery strategy. Moreover, the biological significance of the discovery of in vivo functional small molecules with different biological contexts is also interrogated at a metabolic perspective.

Ureaplasma parvum undergoes selection in utero resulting in genetically diverse isolates colonising the chorioamnion of fetal sheep

Ureaplasmas are the microorganisms most frequently isolated from the amniotic fluid of pregnant women and can cause chronic intrauterine infections. These tiny bacteria are thought to undergo rapid evolution and exhibit a hypermutatable phenotype; however, little is known about how ureaplasmas respond to selective pressures in utero. Using an ovine model of chronic intra-amniotic infection, we investigated if exposure of ureaplasmas to sub-inhibitory concentrations of erythromycin could induce phenotypic or genetic indicators of macrolide resistance. At 55 days gestation, 12 pregnant ewes received an intra-amniotic injection of a non-clonal, clinical U. parvum strain, followed by: (i) erythromycin treatment (IM, 30 mg/kg/day, n=6); or (ii) saline (IM, n=6) at 100 days gestation. Fetuses were then delivered surgically at 125 days gestation. Despite injecting the same inoculum into all ewes, significant differences between amniotic fluid and chorioamnion ureaplasmas were detected following chronic intra-amniotic infection. Numerous polymorphisms were observed in domain V of the 23S rRNA gene of ureaplasmas isolated from the chorioamnion (but not the amniotic fluid), resulting in a mosaic-like sequence. Chorioamnion isolates also harboured the macrolide resistance genes erm(B) and msr(D) and were associated with variable roxithromycin minimum inhibitory concentrations. Remarkably, this variability occurred independently of exposure of ureaplasmas to erythromycin, suggesting that low-level erythromycin exposure does not induce ureaplasmal macrolide resistance in utero. Rather, the significant differences observed between amniotic fluid and chorioamnion ureaplasmas suggest that different anatomical sites may select for ureaplasma sub-types within non-clonal, clinical strains. This may have implications for the treatment of intrauterine ureaplasma infections.

The mouse model of Chlamydia genital tract infection: a review of infection, disease, immunity and vaccine development

Chlamydia trachomatis is the most common sexually transmitted bacterial infection worldwide. The impact of this pathogen on human reproduction has intensified research efforts to better understand chlamydial infection and pathogenesis. Whilst there are animal models available that mimic the many aspects of human chlamydial infection, the mouse is regarded as the most practical and widely used of the models. Studies in mice have greatly contributed to our understanding of the host-pathogen interaction and provided an excellent medium for evaluating vaccines. Here we explore the advantages and disadvantages of all animal models of chlamydial genital tract infection, with a focus on the murine model and what we have learnt from it so far.

Multilocus sequence analysis provides insights into molecular epidemiology of Chlamydia pecorum infections in Australian sheep, cattle and koalas

Chlamydia pecorum is a significant pathogen of domestic livestock and wildlife. We have developed a C. pecorum-specific multilocus sequence analysis (MLSA) scheme to examine the genetic diversity of and relationships between Australian sheep, cattle, and koala isolates. An MLSA of seven concatenated housekeeping gene fragments was performed using 35 isolates, including 18 livestock isolates (11 Australian sheep, one Australian cow, and six U.S. livestock isolates) and 17 Australian koala isolates. Phylogenetic analyses showed that the koala isolates formed a distinct clade, with limited clustering with C. pecorum isolates from Australian sheep. We identified 11 MLSA sequence types (STs) among Australian C. pecorum isolates, 10 of them novel, with koala and sheep sharing at least one identical ST (designated ST2013Aa). ST23, previously identified in global C. pecorum livestock isolates, was observed here in a subset of Australian bovine and sheep isolates. Most notably, ST23 was found in association with multiple disease states and hosts, providing insights into the transmission of this pathogen between livestock hosts. The complexity of the epidemiology of this disease was further highlighted by the observation that at least two examples of sheep were infected with different C. pecorum STs in the eyes and gastrointestinal tract. We have demonstrated the feasibility of our MLSA scheme for understanding the host relationship that exists between Australian C. pecorum strains and provide the first molecular epidemiological data on infections in Australian livestock hosts.

Evolution to a chronic disease niche correlates with increased sensitivity to tryptophan availability for the obligate intracellular bacterium Chlamydia pneumoniae

The chlamydiae are obligate intracellular parasites that have evolved specific interactions with their various hosts and host cell types to ensure their successful survival and consequential pathogenesis. The species Chlamydia pneumoniae is ubiquitous, with serological studies showing that most humans are infected at some stage in their lifetime. While most human infections are asymptomatic, C. pneumoniae can cause more-severe respiratory disease and pneumonia and has been linked to chronic diseases such as asthma, atherosclerosis, and even Alzheimer's disease. The widely dispersed animal-adapted C. pneumoniae strains cause an equally wide range of diseases in their hosts. It is emerging that the ability of C. pneumoniae to survive inside its target cells, including evasion of the host's immune attack mechanisms, is linked to the acquisition of key metabolites. Tryptophan and arginine are key checkpoint compounds in this host-parasite battle. Interestingly, the animal strains of C. pneumoniae have a slightly larger genome, enabling them to cope better with metabolite restrictions. It therefore appears that as the evolutionarily more ancient animal strains have evolved to infect humans, they have selectively become more "susceptible" to the levels of key metabolites, such as tryptophan. While this might initially appear to be a weakness, it allows these human C. pneumoniae strains to exquisitely sense host immune attack and respond by rapidly reverting to a persistent phase. During persistence, they reduce their metabolic levels, halting progression of their developmental cycle, waiting until the hostile external conditions have passed before they reemerge.