A laboratory competency examination in microbiology.

The American Society for Microbiology's curricular guidelines for Introductory Microbiology highlighted key laboratory skills in the isolation, visualization and identification of microorganisms as core learning objectives in the discipline. Since the publication of these guidelines in 2012, there has been a paucity of diagnostic assessment tools in the literature that can be used to assess competencies in the microbiology laboratory. This project aimed to establish a laboratory competency examination for introductory microbiology, with tasks specifically aligned to laboratory skills and learning outcomes outlined in curricular guidelines for microbiology. A Laboratory Competency Examination assessing student skills in light microscopy, Gram-staining, pure culture, aseptic technique, serial dilution, dilution calculations and pipetting was developed at The University of Queensland, Australia. The Laboratory Competency Examination was field-tested in a large introductory microbiology subject (∼400 students), and student performance and learning gains data were collected from 2016 to 2017 to evaluate the validity of the assessment. The resulting laboratory assessment is presented as an endpoint diagnostic tool for assessing laboratory competency that can be readily adapted towards different educational contexts.

Dissecting the apicomplexan rhoptry neck proteins.

Apicomplexan parasites possess specialized secretory organelles (rhoptries and micronemes) that release their contents during host cell invasion. Although the rhoptries were once thought to be merely a bulbous 'protein reservoir' connected to an anterior neck region, the localization of a protein specifically to the neck suggested that this region was more than just a duct. Recent studies have shown that the rhoptry neck sub-compartment possesses a distinct protein repertoire. Some of these proteins share common features, including conservation across the phylum and involvement in tight-junction formation. A sub-group of rhoptry neck proteins, the RONs, their association with the microneme protein apical membrane antigen AMA1, and their involvement in invasion are discussed.

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton.

Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (K(d)=0.60 microM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.

Plasmodium falciparum: genetic and immunogenic characterisation of the rhoptry neck protein PfRON4.

The Apicomplexan parasites Toxoplasma and Plasmodium, respectively, cause toxoplasmosis and malaria in humans and although they invade different host cells they share largely conserved invasion mechanisms. Plasmodium falciparum merozoite invasion of red blood cells results from a series of co-ordinated events that comprise attachment of the merozoite, its re-orientation, release of the contents of the invasion-related apical organelles (the rhoptries and micronemes) followed by active propulsion of the merozoite into the cell via an actin-myosin motor. During this process, a tight junction between the parasite and red blood cell plasma membranes is formed and recent studies have identified rhoptry neck proteins, including PfRON4, that are specifically associated with the tight junction during invasion. Here, we report the structure of the gene that encodes PfRON4 and its apparent limited diversity amongst geographically diverse P. falciparum isolates. We also report that PfRON4 protein sequences elicit immunogenic responses in natural human malaria infections.

Characterisation of PfRON6, a Plasmodium falciparum rhoptry neck protein with a novel cysteine-rich domain.

The pathological consequences of malaria infection are the result of parasite replication within red blood cells (RBCs). Invasion into RBCs is mediated by a large repertoire of parasite proteins that are distributed on the parasite surface and within specialised apical secretory organelles. As invasion is an essential step in the parasite life-cycle, targeting invasion-related molecules provides an avenue for therapeutic intervention. We have used genome and transcriptome data available for Plasmodium falciparum to identify proteins likely to be involved in RBC invasion. Of these candidates, we selected a protein which we have dubbed PfRON6 for detailed characterisation. PfRON6 contains a novel cysteine-rich domain that is conserved in other Apicomplexan parasites. We show that PfRON6 is localised in the rhoptry neck of merozoites and is transferred to the newly formed parasitophorous vacuole during invasion. Transfection experiments indicate that the gene which encodes PfRON6 is refractory to integration that disrupts the coding sequence, suggesting its absence is incompatible with the parasite life-cycle. Further, the cysteine-rich domain appears to be functionally important as it cannot be truncated. Taken together, these data identify PfRON6 as a novel and potentially important component of the Plasmodium invasion machinery.

Plasmodium falciparum purine nucleoside phosphorylase is critical for viability of malaria parasites.

Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Deltapfpnp). Lysates of the Deltapfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Deltapfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Deltapfpnp parasites. The Deltapfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.

Plasmodium falciparum: growth response to potassium channel blocking compounds.

Potassium channels are essential for cell survival and regulate the cell membrane potential and electrochemical gradient. During its lifecycle, Plasmodium falciparum parasites must rapidly adapt to dramatically variant ionic conditions within the mosquito mid-gut, the hepatocyte and red blood cell (RBC) cytosols, and the human circulatory system. To probe the participation of K(+) channels in parasite viability, growth response assays were performed in which asexual stage P. falciparum parasites were cultured in the presence of various Ca(2+)-activated K(+) channel blocking compounds. These data describe the novel anti-malarial effects of bicuculline methiodide and tubocurarine chloride and the novel lack of effect of apamine and verruculogen. Taken together, the data herein imply the presence of K(+) channels, or other parasite-specific targets, in P. falciparum-infected RBCs that are sensitive to blockade with Ca(2+)-activated K(+) channel blocking compounds.

Characterization of two putative potassium channels in Plasmodium falciparum.

BACKGROUND: Potassium channels are essential for cell survival and participate in the regulation of cell membrane potential and electrochemical gradients. During its lifecycle, Plasmodium falciparum parasites must successfully traverse widely diverse environmental milieus, in which K+ channel function is likely to be critical. Dramatically differing conditions will be presented to the parasite in the mosquito mid-gut, red blood cell (RBC) cytosol and the human circulatory system. METHODS: In silico sequence analyses identified two open-reading frames in the P. falciparum genome that are predicted to encode for proteins with high homology to K+ channels. To further analyse these putative channels, specific antisera were generated and used in immunoblot and immunofluorescence analyses of P. falciparum-infected RBCs. Recombinant genome methods in cultured P. falciparum were used to create genetic knock outs of each K+ channel gene to asses the importance of their expression. RESULTS: Immunoblot and IFA analyses confirmed the expression of the two putative P. falciparum K+ channels (PfK1 and PfK2). PfK1 is expressed in all asexual stage parasites, predominantly in late stages and localizes to the RBC membrane. Conversely, PfK2 is predominantly expressed in late schizont and merozoite stage parasites and remains primarily localized to the parasite. Repeated attempts to knockout PfK1 and PfK2 expression by targeted gene disruption proved unsuccessful despite evidence of recombinant gene integration, indicating that pfk1 and pfk2 are apparently refractory to genetic disruption. CONCLUSION: Putative K+ channel proteins PfK1 and PfK2 are expressed in cultured P. falciparum parasites with differing spatial and temporal patterns. Eventual functional characterization of these channels may reveal future pharmacological targets.

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton.

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.

Plasmodium falciparum Pf34, a novel GPI-anchored rhoptry protein found in detergent-resistant microdomains.

Apicomplexan parasites are characterised by the presence of specialised organelles, such as rhoptries, located at the apical end of invasive forms that play an important role in invasion of the host cell and formation of the parasitophorous vacuole. In this study, we have characterised a novel Plasmodium falciparum rhoptry protein, Pf34, encoded by a single exon gene located on chromosome 4 and expressed as a 34kDa protein in mature asexual stage parasites. Pf34 is expressed later in the life cycle than the previously described rhoptry protein, Rhoptry Associated Membrane Antigen (RAMA). Orthologues of Pf34 are present in other Plasmodium species and a potential orthologue has also been identified in Toxoplasma gondii. Indirect immunofluorescence assays show that Pf34 is located at the merozoite apex and localises to the rhoptry neck. Pf34, previously demonstrated to be glycosyl-phosphatidyl-inositol (GPI)-anchored [Gilson, P.R., Nebl, T., Vukcevic, D., Moritz, R.L., Sargeant, T., Speed, T.P., Schofield, L., Crabb, B.S. (2006) Identification and stoichiometry of GPI-anchored membrane proteins of the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 5, 1286-1299.], is associated with parasite-derived detergent-resistant microdomains (DRMs). Pf34 is carried into the newly invaded ring, consistent with a role for Pf34 in the formation of the parasitophorous vacuole. Pf34 is exposed to the human immune system during infection and is recognised by human immune sera collected from residents of malaria endemic areas of Vietnam and Papua New Guinea.

Chloroquine resistance modulated in vitro by expression levels of the Plasmodium falciparum chloroquine resistance transporter.

Plasmodium falciparum malaria is increasingly difficult to treat and control due to the emergence of parasite resistance to the major antimalarials, notably chloroquine. Recent work has shown that the chloroquine resistance phenotype can be conferred by multiple amino acid mutations in the parasite digestive vacuole transmembrane protein PfCRT. Here, we have addressed whether chloroquine resistance can also be affected by changes in expression levels of this protein. Transient transfection reporter assays revealed that truncation of the pfcrt 3'-untranslated region just prior to putative polyadenylation sites resulted in a 10-fold decrease in luciferase expression levels. Using allelic exchange on a chloroquine-resistant line (7G8 from Brazil), this truncated 3'-untranslated region was inserted downstream of the pfcrt coding sequence, in the place of the endogenous 3'-untranslated region. The resulting pfcrt-modified "knockdown" clones displayed a marked decrease in pfcrt transcription and an estimated 30-40% decrease in PfCRT protein expression levels. [3H]hypoxanthine incorporation assays demonstrated up to a 40% decrease in chloroquine with or without verapamil IC50 levels of pfcrt knockdown clones, relative to the 7G8 parent. Single-cell photometric analyses were consistent with an altered intracellular pH in the knockdown clones, providing further evidence for a relationship between PfCRT, pH regulation, and chloroquine resistance. Genetic truncation of 3'-untranslated regions provides a useful approach for assessing the impact of candidate genes on drug resistance or other quantifiable phenotypes in P. falciparum.

Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells.

The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria.