Correlation of antifungal susceptibility and sequence types within Cryptococcus neoformans VNI from HIV patients, and ERG11 gene polymorphism.

INTRODUCTION: Here we tested the correlation between minimum inhibitory concentrations (MICs) of major antifungal agents and sequence types (STs) within Cryptococcus neoformans VNI isolates, and explored the ERG11 gene of included strains. MATERIALS AND METHODS: We analysed 23 C. neoformans strains categorised into two groups according to the distribution of the ST profile in Kinshasa clinics (Democratic Republic of Congo): major ST [ST93 (n = 15)], and less common STs [ST659 (n = 2), ST5 (n = 2), ST4 (n = 1), ST 53 (n = 1), ST31 (n = 1), and ST69 (n = 1)]. The MICs of the major antifungal agents [amphotericin B (AMB), 5-fluorocytosine (5FC) and fluconazole (FCZ)] were determined following EUCAST guidelines. ERG11 gene sequences were extracted from whole genome sequence of the isolates and compared with the wild-type gene sequence of the C. neoformans VNI. RESULTS: Although major ST isolates appeared to have lower median MICs for AMB and 5FU than less common ST isolates (0.50 vs. 0.75 mg/L for AMB, 2 vs. 4 mg/L for 5FU, respectively), FCZ susceptibility was similar in both groups (4 mg/L) (p-value >0.05). The susceptibility profile of C. neoformans strains separately considered did not significantly affect the patients' clinical outcomes (p-value >0.05). Furthermore, two structural modalities of the ERG11 gene were observed: (1) that of the reference gene, and (2) that containing two exonic silent point substitutions, and one intronic point substitution located in a sequence potentially involved in pre-mRNA splicing (c.337-22C > T); with no association with the MICs of the isolates (p-value >0.05). CONCLUSIONS: The lack of association/correlation found in this study calls for further investigations to better understand the mechanisms of C. neoformans resistance to antifungal agents.

Clinical epidemiology and high genetic diversity amongst Cryptococcus spp. isolates infecting people living with HIV in Kinshasa, Democratic Republic of Congo.

Neuromeningeal cryptococcosis (NMC) is a life-threatening opportunistic infection in advanced HIV disease patients (AHDP). It is caused by Cryptococcus spp. complexes and mainly occurs in sub-Saharan Africa. In this study, we performed molecular characterization and antifungal susceptibility profiling of Cryptococcus isolates from AHDP in Kinshasa (DRC). Additionally, we investigated a possible association between NMC severity factors and the Cryptococcus neoformans (Cn) multilocus sequence typing (MLST) profiles. We characterized the isolates using PCR serotyping, MALDI-TOF MS, internal transcribed spacer (ITS) sequencing, and MLST. Susceptibility testing for the major antifungal drugs was performed according to the EUCAST guidelines. Parameters associated with NMC severity, such as hypoglycorrhachia (< 50 mg/dL), increased cerebral spinal fluid opening pressure (> 30 cm H2O), and poor therapeutic outcome were compared with the Cn MLST sequences type (ST). Twenty-three out of 29 Cryptococcus isolates were identified as serotype A using PCR serotyping (79.3%; 95% IC: 65.5-93.1), while six (20.7%; 95% IC: 6.9-34.5) were not serotypable. The 29 isolates were identified by ITS sequencing as follows: Cryptococcus neoformans (23/29, 79.3%), Cutaneotrichosporon curvatus (previously called Cryptococcus curvatus) (5/29, 17.2%), and Papiliotrema laurentii (Cryptococcus laurentii) (1/29, 3.5%). Using the ISHAM MLST scheme, all Cn isolates were identified as molecular type VNI. These comprised seven different STs: ST93 (n = 15), ST5 (n = 2), ST53 (n = 1), ST31 (n = 1), ST4 (n = 1), ST69 (n = 1), and one novel ST that has not yet been reported from other parts of the world and was subsequently assigned as ST659 (n = 2). Of the included strains, only Papiliotrema laurentii was resistant to amphoterin B (1/29, 3.5%), 6.8% (2/29) were resistant to 5-flucytosine (the single Papiliotrema laurentii strain and one Cryptococcus neoformans isolate), and 13.8% (4/29) to fluconazole, including two of five (40%) Cutaneotrichosporon curvatus and two of 23 (8.7%) C. neoformans strains. We found a significative association between poor therapeutic outcome and a non-ST93 sequence type of causative strains (these concerned the less common sequence types: ST53, ST31, ST5, ST4, ST659, and ST69) (87.5% versus 40%, p = 0.02). Molecular analysis of Cryptococcus spp. isolates showed a wide species diversity and genetic heterogenicity of Cn within the VNI molecular type. Furthermore, it is worrying that among included strains we found resistances to several of the commonly used antifungals.

Structure and activity of the DHNA Coenzyme-A Thioesterase from Staphylococcus aureus providing insights for innovative drug development.

Humanity is facing an increasing health threat caused by a variety of multidrug resistant bacteria. Within this scenario, Staphylococcus aureus, in particular methicillin resistant S. aureus (MRSA), is responsible for a number of hospital-acquired bacterial infections. The emergence of microbial antibiotic resistance urgently requires the identification of new and innovative strategies to treat antibiotic resistant microorganisms. In this context, structure and function analysis of potential drug targets in metabolic pathways vital for bacteria endurance, such as the vitamin K2 synthesis pathway, becomes interesting. We have solved and refined the crystal structure of the S. aureus DHNA thioesterase (SaDHNA), a key enzyme in the vitamin K2 pathway. The crystallographic structure in combination with small angle X-ray solution scattering data revealed a functional tetramer of SaDHNA. Complementary activity assays of SaDHNA indicated a preference for hydrolysing long acyl chains. Site-directed mutagenesis of SaDHNA confirmed the functional importance of Asp16 and Glu31 for thioesterase activity and substrate binding at the putative active site, respectively. Docking studies were performed and rational designed peptides were synthesized and tested for SaDHNA inhibition activity. The high-resolution structure of SaDHNA and complementary information about substrate binding will support future drug discovery and design investigations to inhibit the vitamin K2 synthesis pathway.

Protein-crystal detection with a compact multimodal multiphoton microscope.

There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future.

Pulsed electric fields induce modulation of protein liquid-liquid phase separation.

The time-resolved dynamic assembly and the structures of protein liquid dense clusters (LDCs) were analyzed under pulsed electric fields (EFs) applying complementary polarized and depolarized dynamic light scattering (DLS/DDLS), optical microscopy, and transmission electron microscopy (TEM). We discovered that pulsed EFs substantially affected overall morphologies and spatial distributions of protein LDCs and microcrystals, and affected the phase diagrams of LDC formation, including enabling protein solutions to overcome the diffusive flux energy barrier to phase separate. Data obtained from DLS/DDLS and TEM showed that LDCs appeared as precursors of protein crystal nuclei, followed by the formation of ordered structures within LDCs applying a pulsed EF. Experimental results of circular dichroism spectroscopy provided evidence that the protein secondary structure content is changing under EFs, which may consequently modulate protein-protein interactions, and the morphology, dimensions, and internal structure of LDCs. Data and results obtained unveil options to modulate the phase diagram of crystallization, and physical morphologies of protein LDCs and microcrystals by irradiating sample suspensions with pulsed EFs.

Solution Structures and Dynamic Assembly of the 24-Meric Plasmodial Pdx1-Pdx2 Complex.

Plasmodium species are protozoan parasites causing the deadly malaria disease. They have developed effective resistance mechanisms against most antimalarial medication, causing an urgent need to identify new antimalarial drug targets. Ideally, new drugs would be generated to specifically target the parasite with minimal or no toxicity to humans, requiring these drug targets to be distinctly different from the host's metabolic processes or even absent in the host. In this context, the essential presence of vitamin B6 biosynthesis enzymes in Plasmodium, the pyridoxal phosphate (PLP) biosynthesis enzyme complex, and its absence in humans is recognized as a potential drug target. To characterize the PLP enzyme complex in terms of initial drug discovery investigations, we performed structural analysis of the Plasmodium vivax PLP synthase domain (Pdx1), glutaminase domain (Pdx2), and Pdx1-Pdx2 (Pdx) complex (PLP synthase complex) by utilizing complementary bioanalytical techniques, such as dynamic light scattering (DLS), X-ray solution scattering (SAXS), and electron microscopy (EM). Our investigations revealed a dodecameric Pdx1 and a monodispersed Pdx complex. Pdx2 was identified in monomeric and in different oligomeric states in solution. Interestingly, mixing oligomeric and polydisperse Pdx2 with dodecameric monodisperse Pdx1 resulted in a monodispersed Pdx complex. SAXS measurements revealed the low-resolution dodecameric structure of Pdx1, different oligomeric structures for Pdx2, and a ring-shaped dodecameric Pdx1 decorated with Pdx2, forming a heteromeric 24-meric Pdx complex.

Protein phase separation and determinants of in cell crystallization.

Liquid-liquid phase separation (LLPS) in cells is known as a complex physicochemical process causing the formation of membrane-less organelles (MLOs). Cells have well-defined different membrane-surrounded organelles like mitochondria, endoplasmic reticulum, lysosomes, peroxisomes, etc., however, on demand they can create MLOs as stress granules, nucleoli and P bodies to cover vital functions and regulatory activities. However, the mechanism of intracellular molecule assembly into functional compartments within a living cell remains till now not fully understood. in vitro and in vivo investigations unveiled that MLOs emerge after preceding liquid-liquid, liquid-gel, liquid-semi-crystalline, or liquid-crystalline phase separations. Liquid-liquid and liquid-gel MLOs form the majority of cellular phase separation events, while the occurrence of micro-sized crystals in cells was only rarely observed, however can be considered as a result of a preceding protein phase separation event. In vivo, also known and termed as in cellulo crystals, are reported since 1853. In some cases, they have been linked to vital cellular functions, such as storage and detoxification. However, the occurrence of in cellulo crystals is also associated to diseases like cataract, hemoglobin C diseases, etc. Therefore, better knowledge about the involved molecular processes will support drug discovery investigations to cure diseases related to in cellulo crystallization. We summarize physical and chemical determinants known today required for phase separation initiation and formation and in cellulo crystal growth. In recent years it has been demonstrated that LLPS plays a crucial role in cell compartmentalization and formation of MLOs. Here we discuss potential mechanisms and potential crowding agents involved in protein phase separation and in cellulo crystallization.

The conserved hypothetical protein Tb427.10.13790 is required for cytokinesis in Trypanosoma brucei.

Trypanosoma brucei, a flagellated protozoan causing the deadly tropical disease Human African Trypanosomiasis (HAT), affects people in sub-Saharan Africa. HAT therapy relies upon drugs which use is limited by toxicity and rigorous treatment regimes, while development of vaccines remains elusive, due to the effectiveness of the parasite´s antigenic variation. Here, we evaluate a hypothetical protein Tb427.10.13790, as a potential drug target. This protein is conserved among all kinetoplastids, but lacks homologs in all other pro- and eukaryotes. Knockdown of Tb427.10.13790 resulted in appearance of monster cells containing multiple nuclei and multiple flagella, a considerable enlargement of the flagellar pocket and eventually a lethal phenotype. Furthermore, analysis of kinetoplast and nucleus division in the knockdown cell line revealed a partial cell cycle arrest and failure to initiate cytokinesis. Likewise, overexpression of the respective protein fused with enhanced green fluorescent protein was also lethal for T. brucei. In these cells, the labelled protein appeared as a single dot near kinetoplast and flagellar pocket. Our results reveal that Tb427.10.13790 is essential for the parasite´s viability and may be a suitable new anti-trypanosomatid drug target candidate. Furthermore, we suggest that it might be worthwhile to investigate also other of the many so far just annotated trypanosome genes as a considerable number of them to lack human homologs but may be of critical importance for the kinetoplastid parasites.

African trypanosomes and brain infection - the unsolved question.

African trypanosomes induce sleeping sickness. The parasites are transmitted during the blood meal of a tsetse fly and appear primarily in blood and lymph vessels, before they enter the central nervous system. During the latter stage, trypanosomes induce a deregulation of sleep-wake cycles and some additional neurological disorders. Historically, it was assumed that trypanosomes cross the blood-brain barrier and settle somewhere between the brain cells. The brain, however, is a strictly controlled and immune-privileged area that is completely surrounded by a dense barrier that covers the blood vessels: this is the blood-brain barrier. It is known that some immune cells are able to cross this barrier, but this requires a sophisticated mechanism and highly specific cell-cell interactions that have not been observed for trypanosomes within the mammalian host. Interestingly, trypanosomes injected directly into the brain parenchyma did not induce an infection. Likewise, after an intraperitoneal infection of rats, Trypanosoma brucei brucei was not observed within the brain, but appeared readily within the cerebrospinal fluid (CSF) and the meninges. Therefore, the parasite did not cross the blood-brain barrier, but the blood-CSF barrier, which is formed by the choroid plexus, i.e. the part of the ventricles where CSF is produced from blood. While there is no question that trypanosomes are able to invade the brain to induce a deadly encephalopathy, controversy exists about the pathway involved. This review lists experimental results that support crossing of the blood-brain barrier and of the blood-CSF barrier and discuss the implications that either pathway would have on infection progress and on the survival strategy of the parasite. For reasons discussed below, we prefer the latter pathway and suggest the existence of an additional distinct meningeal stage, from which trypanosomes could invade the brain via the Virchow-Robin space thereby bypassing the blood-brain barrier. We also consider healthy carriers, i.e. people living symptomless with the disease for up to several decades, and discuss implications the proposed meningeal stage would have for new anti-trypanosomal drug development. Considering the re-infection of blood, a process called relapse, we discuss the likely involvement of the newly described glymphatic connection between the meningeal space and the lymphatic system, that seems also be important for other infectious diseases.

In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins.

During the last decade, the number of three-dimensional structures solved by X-ray crystallography has increased dramatically. By 2014, it had crossed the landmark of 100 000 biomolecular structures deposited in the Protein Data Bank. This tremendous increase in successfully crystallized proteins is primarily owing to improvements in cloning strategies, the automation of the crystallization process and new innovative approaches to monitor crystallization. However, these improvements are mainly restricted to soluble proteins, while the crystallization and structural analysis of membrane proteins or proteins that undergo major post-translational modifications remains challenging. In addition, the need for relatively large crystals for conventional X-ray crystallography usually prevents the analysis of dynamic processes within cells. Thus, the advent of high-brilliance synchrotron and X-ray free-electron laser (XFEL) sources and the establishment of serial crystallography (SFX) have opened new avenues in structural analysis using crystals that were formerly unusable. The successful structure elucidation of cathepsin B, accomplished by the use of microcrystals obtained by in vivo crystallization in baculovirus-infected Sf9 insect cells, clearly proved that crystals grown intracellularly are very well suited for X-ray analysis. Here, methods by which in vivo crystals can be obtained, isolated and used for structural analysis by novel highly brilliant XFEL and synchrotron-radiation sources are summarized and discussed.