Biology subjects

Phan, T. H.

Publications and source records attributed to Phan, T. H..

4 recordsLinked to original sources

bioRxiv2021

Nanocharacterisation of extracellular vesicles uncovers the correlation between isolation methods, physicochemical composition and their biological function

Extracellular vesicles (EVs) have been lauded as next generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass-produce EVs, i.e. to isolate, purify and characterise them effectively. Technical limitations in comprehensive characterisation of EVs leads to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were shown to correlate well with the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterisation approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

cell biology↗

bioRxiv2020

Rerouting of an Esx substrate pair from the ESX-1 type VII secretion system to ESX-5 by modifying a PE/PPE substrate pair

Type VII secretion systems (T7SSs) secrete a wide range of extracellular proteins that play important roles in bacterial viability and in host-pathogen interactions of pathogenic mycobacteria. There are five subtypes of mycobacterial T7SSs, called ESX-1 to ESX-5, and four classes of T7SS substrates, namely the Esx, PE, PPE and Esp proteins. At least some of these substrates are secreted as heterodimers. The ESX systems mediate the secretion of specific members of the Esx, PE and PPE proteins, raising the question how these substrates are recognized in a system-specific fashion. PE/PPE heterodimers interact with their cognate EspG chaperones, which recently has been shown to determine their designated secretion pathway. Both structural and pulldown analysis suggest that EspG is unable to interact with Esx proteins and therefore the determining factor for system-specificity of these substrates remains unknown. In this study, we have investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 (MMAR_0187/MMAR _0188) in Mycobacterium marinum. While this substrate pair was hardly secreted when ectopically expressed, secretion was observed when EsxB_1/EsxA_1 was co-expressed together with PE35/PPE68_1 (MMAR_0185/MMAR_0186), which are encoded by the same operon. Surprisingly, co-expressing EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in co-secretion of EsxB_1/EsxA_1 via ESX-5. Our data suggest a secretion model in which PE35/PPE68_1 is a determinant factor for the system-specific secretion of EsxB_1/EsxA_1.

molecular biology↗

bioRxiv2017

MULTILOCUS SEQUENCE TYPING REVEALS A UNIQUE CO-DOMINANT POPULATION STRUCTURE OF CRYPTOCOCCUS NEOFORMANS VAR. GRUBII IN VIETNAM

Cryptococcosis is amongst the most important invasive fungal infections globally, with cryptococcal meningitis causing an estimated 180,000 deaths each year in HIV infected patients alone. Patients with other forms of immunosuppression are also at risk, and disease is increasingly recognized in apparently immunocompetent individuals. Cryptococcus neoformans var. grubii (serotype A, molecular type VNI) has a global distribution and is responsible for the majority of cases. Here, we used the consensus ISHAM Multilocus Sequence Typing (MLST) for C. neoformans to define the population structure of clinical isolates of Cryptococcus neoformans var. grubii from Vietnam (n=136) and Laos (n=81). We placed these isolates into the global context using published MLST data from 8 other countries (total N = 669). We observed a phylo-geographical relationship in which Laos was similar to its Southeast Asian neighbor Thailand in being dominated (83%) by Sequence Type (ST) 4 and its Single Locus Variant ST6. On the other hand, Vietnam was uniquely intermediate between Southeast Asia and East Asia having both ST4/ST6 (35%) and ST5 (48%) which causes the majority of cases in East Asia. Analysis of genetic distance (Fst) between different populations of Cryptococcus neoformans var. grubii supported the intermediate nature of the population from Vietnam. A strong association between ST5 and infection in apparently immunocompetent, HIV-uninfected patients was observed in Vietnam (OR: 7.97, [95%CI: 3.18-19.97], p < 0.0001). Our study emphasizes that Vietnam, with its intermediate Cryptococcus neoformans var. grubii population structure, provides the strongest epidemiological evidence of the relationship between ST5 and infection of HIV-uninfected patients. Human population genetic distances within the region suggest these differences in CNVG population across Southeast Asia are driven by ecological factors rather than host factors.\n\nAuthor summaryCryptococcus neoformans is a yeast that causes meningitis in people, usually with damaged immune systems. There are >180,000 deaths in HIV-infected patients each year, most occurring where there are the highest HIV/AIDS disease burdens. Vietnam and Laos have contributed significantly to clinical trials aiming to improve the treatment of cryptococcal meningitis, but the relationship of isolates from these countries to the global population is not yet described. Here, we address this knowledge gap by using Multilocus Sequence Typing to study the population of Cryptococcus neoformans var. grubii (CNVG) in Laos and Vietnam, with the specific aim of incorporating these populations into the wider global context. We found that, in most countries, a single lineage (family) of strains was responsible for most disease. The Vietnamese CNVG population was unusual in that 2 main lineages circulated at the same time. The Vietnamese CNVG population occupies a middle ground between Thailand/Laos in the west and China in the east. The differences in population structure moving from West to East are probably due to ecological differences. Disease in HIV uninfected patients was almost always due to members of a single family of strains (ST5).

molecular biology↗

bioRxiv2017

N VITRO AND IN VIVO CHARACTERISATION OF ISOLATES OF CRYPTOCOCCUS NEOFORMANS CAUSING MENINGITIS IN HIV-INFECTED AND UNINFECTED PATIENTS IN VIETNAM

We previously observed a substantial burden of cryptococcal meningitis in Vietnam atypically arising in HIV-uninfected individuals. This disease was associated with a single genotype of Cryptococcus neoformans (Sequence Type (ST)5), which was significantly less common in HIV-infected individuals. Aiming to compare the phenotypic characteristics of ST5 and non-ST5 C. neoformans we selected 30 representative Vietnamese isolates, compared their in vitro pathogenic potential and in vivo virulence. ST5 and non-ST5 organisms exhibited comparable characteristics with respect to in vitro virulence markers including melanin production, replication at 37{degrees}C, and growth in cerebrospinal fluid. However, the ST5 isolates had significantly increased variability in cellular and capsular sizing compared with non-ST5 organisms (p<0.001). Counter-intuitively, mice infected with ST5 isolates had significantly longer survival with lower fungal burdens at day 7 than non-ST5 isolates. Notably, ST5 isolates induced significantly greater initial inflammatory responses than non-ST5 strains, measured by TNF- concentrations (p<0.001). Despite being generally less virulent in the mouse model, we hypothesize that the significant within strain variation seen in ST5 isolates in the tested phenotypes may represent an evolutionary advantage enabling adaptation to novel niches including apparently immunocompetent human hosts.

microbiology↗