Welcome to the IUMS 2022 Interactive Program

Background and Aims

Prolidase (E.C 3.4.13.9) is an enzyme that specifically hydrolyzes Xaa-Pro dipeptides into free amino acids. We studied the kinetic behaviours and solved the crystal structure of Lactococcus lactis prolidase (Llprol), showing that this homodimeric enzyme has unique characteristics: allosteric behaviour and substrate inhibition. Mutagenesis studies revealed that the allosteric behaviour of Llprol was eliminated in Llprol D36S and R293S mutants, and substrate inhibition was not detected in Llprol H38S and R293S mutants.

Methods

We solved the crystal structures of D36S, H38S, and R293S Llprol mutants with X-ray crystallography.

Results

Structural comparison between the mutants suggests that Llprol can adapt to different conformational states with distinct substrate affinities, and various interactions between the monomers are the keys to separating these conformational states. We speculate that in allosteric Llprol (WT and H38S), the domain movements required by productive substrate binding are restrained. In addition, we also identified a secondary binding site in Llprol variants that show substrate inhibition. The secondary binding site shares two amino acid residuals with the productive substrate-binding site of Llprol.

Conclusions

In conclusion, we proposed that the relative positioning of monomers and the resulting variation in the structural rearrangements levels and the completeness of certain conformational changes upon substrate binding lead to the allosteric behaviour of Llprol. The substrate inhibition is caused by the presence of a secondary binding site that is partially overlapped with the active site. The damaged secondary binding site explains the removal of substrate inhibition in Llprol H38S and R293S.

Background and Aims

Mounting evidence suggests that the Epstein-Barr virus (EBV), found to be present in intestines of inflammatory bowel disease (IBD) patients, complicates the clinical course of the disease. Studies by our group have shown that EBV DNA increases the production of the inflammatory cytokine IL-17A through Toll-like receptor (TLR) 7 and 9 activation and that it exacerbates inflammation and disease severity in a mouse model of IBD. Hence, the aim of this study was to determine whether TLR7 and 9 inhibition ameliorates the severity of EBV DNA-exacerbated intestinal inflammation in the IBD mouse model.

Methods

Dextran sulfate sodium (DSS)-drinking C57BL/6J mice, the acute colitis model of IBD, were intra-rectally administered the viral DNA and given an intra-peritoneal injection of TLR7 and TLR9 inhibitors. The severity of colitis and inflammation was then evaluated.

Results

Both groups that were administered DSS-drinking water and received intra-rectal EBV DNA along with intra-peritoneal injections of TLR7 or 9 inhibitors had an ameliorated severity of intestinal inflammation when compared to DSS-treated groups that received the viral DNA but not the inhibitors. These groups that were treated with the TLR7 or 9 inhibitors had longer colon lengths, lower histological damage scores, and a lower disease activity index (DAI) score. Immunofluorescence assays also showed lowered numbers of inflammatory cells.

Conclusions

TLR 7 and 9 inhibitors were shown to ameliorate the severity of inflammation in the EBV DNA-exacerbated mouse model of IBD. This study uncovers a potential therapeutic/prophylactic target for IBD management in EBV-infected individuals.

Background and Aims

Mosquitoes are the major vector of arboviruses particularly, dengue virus (DENV), zika virus (ZIKV), and chikungunya virus (CHIKV). Regardless of the failures of current arboviral vector control and prevention strategies for their transmission, alternative approaches are necessitated to further develop and improve. Recent studies have been indicated the important roles of insect-specific viruses (ISVs) in the alteration of the vector competency, serving as alternative schemes for innovative arboviral transmission control. Northeastern (NE) Thailand has a tropical climate that provides opportune settings for mosquitoes’ emergence and dissemination, supporting existing arboviruses epidemics. However, evidence concerning the representative ISVs in potential mosquito vectors of this region is lacking.

Methods

Therefore, this study aimed to survey the burden of ISVs harbored by natural mosquitoes using a Next-generation sequencing (NGS)-based metagenomic approach.

Results

Our analyses reveal that matched viral reads were highly diverse and varied in abundance among mosquito species, which were 11 taxonomic families and an unclassified group. The ISVs reads have prevailed in all mosquito species. Among these, the Guadeloupe mosquito virus (GMV) was stably found in female adults of Aedes aegypti and Culex spp. Notably, the presence of GMV contigs has firstly been suggested as a new spatial distribution of such ISV in NE Thailand as exploration with the public databases of viral metagenomic of Aedes and Culex mosquitoes.

Conclusions

Overall, this study imparts a comprehensive literacy of diverse ISV burdens circulated in the mosquito vectors from NE Thailand, which might be a potential source for innovative strategies of arboviral transmission control.

Background and Aims

Mycetoma is a Neglected Tropical Disease characterized by large subcutaneous swellings and the formation of grains. Mycetoma is treated with itraconazole and surgery with low success rates, resulting in amputations and social sigma. To improve the therapeutic success rates a novel drug is needed. Due to the lack of interest of pharmaceutical industry, the open source drug discovery program mycetOS was established.

Methods

In total 1360 compounds were screened for in vitro activity against M. mycetomatis. Compounds able to inhibit growth at 100 µM, 25 µM, and had an IC50 < 8 µM were selected for studying in vivo efficacy in our M. mycetomatis grain model in the invertebrate Galleria mellonella.

Results

In total 302 compounds were able to inhibit growth at 100 µM and 23 of those met all criteria to be screened in vivo. Of these 23, nine did prolong larval survival. These included 3 out of 7 azoles tested, olorofim, fenbendazole, MMV006357, MMV022478, MMV675968 and MMV1782387. Based on these results, 6 compound series were selected for further studying: fenarimols; aminothiazoles; phenotiazines; dihydrofolate reductase inhibitors; benzimidazoles and ketoximines. For the fenarimols in total 185 additional compounds were screened. By analyzing the in vitro activity and in vivo efficacy in relation to the chemical properties of the fenarimol it appears that the LogD value of a compound was important for in vivo efficacy.

Conclusions

Here we demonstrated that an open source drug discovery project such as MycetOS can be an effective way to identify novel lead compounds for fungal skinNTDs.

Background and Aims

The emergence of new pandemic strains of the MERS-CoV coronavirus in camels necessitates regular monitoring of viral populations in this animal. The goal was to identify viruses circulating among camels in Kazakhstan that pose a potential threat to human and animal health.

Methods

Molecular-genetic and serological methods were used: the isolation of nucleic acids from the camel samples, the construction of libraries for mass parallel sequencing, bioinformatic data analysis, and serological tests.

Results

As a result of virome sequencing, contigs of viruses representing the following families were found: Flaviviridae, Circoviridae, Picobirnaviridae, Astroviridae, Parvoviridae and Hepeviridae. Of these families, all but Astroviruses and Pestiviruses have previously been found in camels. Of particular interest is the detection of short sequences, hepatitis-like viral contigs, which is potentially dangerous for humans. Serological test of camels confirmed the presence of antibodies to the hepatitis E virus, which indicates its possible circulation in camels in Kazakhstan.

Conclusions

The study of viral metagenomes of camels in the close to the Middle East region is an important epidemiological task. This will make it possible to identify not only strains of coronaviruses, but also other unknown viral pathogens that are important both for domestic animals and for human health.

Background and Aims

Salmonella Typhimurium(STM) resides within a modified membrane-bound compartment called Salmonella containing vacuole(SCV) inside the macrophages. The biogenesis and stability of SCV are crucial for the intracellular proliferation of Salmonella. Our research aims to provide a novel mechanism behind the role of Salmonella OmpA in maintaining the stability of SCV.

Methods

Immunofluorescence, Intracellular Proliferation, Texas Red-Ovalbumin-Pulse-Chase Experiment, Acid-Phosphatase assay, Site-directed-mutagenesis, Immunoblotting, Flow cytometry

Results

The OmpA deletion compelled STM to exit the SCV during the early stage of infection. STMΔompA failed to retain LAMP-1 and evaded SCV. The cytosolic STMΔompA population activated the autophagy machinery after colocalizing with syntaxin17 and LC3B. Subsequently, the autophagosomes harboring STMΔompA were targeted to the lysosomes for degradation. Inhibition of the autophagy pathway using bafilomycinA1 restored the intracellular proliferation of STMΔompA. Furthermore, the four extracellular loops of OmpA played a crucial role in maintaining the LAMP-1 pool around the SCV. Upon alteration of the extracellular loop sequences of Salmonella OmpA by site-directed-mutagenesis, STM failed to preserve the interaction between LAMP-1 and the SCV and eventually escaped into the cytosol. STMΔompA and the extracellular loop mutants showed increased recruitment of p62 and LC3B in comparison to the wildtype-STM upon vacuole evasion into the cytosol. Surprisingly, the cytosolic population of Salmonella having mutations in the extracellular loops of OmpA did not activate the lysosomal degradation pathway like STMΔompA, which helped them to survive within the murine macrophages.

Conclusions

Our study revealed an OmpA dependent novel strategy utilized by Salmonella to combat host autophagy by maintaining the stability of SCV.

Background and Aims

Alterations in the gut microbiota have been associated with Celiac Disease (CeD); however, the contribution of individual microbial species to the overall functional activity of the microbiota (i.e., the gut metabolome) and their role in CeD pathogenesis are largely unknown. This is because existing functional profiling approaches merely enumerate functional pathways encoded by metagenomes but do not provide any information about what microbes carry what pathways or produce what metabolites. To address this gap, we leveraged data from a perspective birth cohort study of infants at high risk of CeD to functionally profile their gut microbiota at species and molecular level resolution.

Methods

We used 167 fecal metagenomes collected longitudinally from ten children who developed CeD and ten controls to construct genome-scale models of metabolism for 359 microbial species in these samples. The models for species present in each sample were then integrated to build sample-specific species-resolved models of the entire gut microbiota metabolism.

Results

By computationally simulating these models, we could track back individual microbial species producing specific secreted metabolites. We identified 18 (out of 2,120) species-metabolite pairs involving nine species and 14 metabolites, that were significantly different between cases and controls (Wilcox, p < 0.05). Some of these metabolites were previously implicated in CeD or inflammation, e.g., L-tryptophan, arabinose, xanthine, and cholic acid that are linked to species of Bacteroides, Bifidobacterium, and Blautia in our models.

Conclusions

Overall, this study provides a roadmap for mechanistically linking microbial and molecular markers of CeD in the gut.

Background and Aims

Foot-and-mouth disease virus (FMDV) and classical swine fever virus (CSFV) possess positive-sense single-stranded RNA genomes and an internal ribosomal entry site (IRES) element within their 5¢-untranslated regions.

Methods

To investigate the common host factors associated with these IRESs, we established cell lines expressing a bicistronic luciferase reporter plasmid containing an FMDV-IRES or CSFV-IRES element between the Renilla and firefly luciferase genes.

Results

First, we treated FMDV-IRES cells with the French maritime pine extract, Pycnogenol (PYC), and examined its suppressive effect on FMDV-IRES activity, as PYC has been reported to have antiviral properties. Next, we performed microarray analysis to identify the host factors that modified their expression upon treatment with PYC, and confirmed their function using specific siRNAs. We found that polycystic kidney disease 1-like 3 (PKD1L3) and ubiquitin-specific peptidase 31 (USP31) were associated with FMDV-IRES activity. Moreover, silencing of these factors significantly suppressed CSFV-IRES activity.

Conclusions

Thus, PKD1L3 and USP31 are host factors associated with the functions of FMDV- and CSFV-IRES elements.

Background and Aims

Orthohantaviruses are emerging zoonotic viruses with rodents as their main reservoir. Upon respiratory transmission, human infection can result in hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) depending on the causative virus. Endothelial cells are predominantly infected with endothelial cell activation and increased vascular permeability likely to play a central role in pathogenesis. Although microvasculature of multiple organs can become infected, distinct clinical outcomes between HFRS and HCPS exist. Endothelial cells cultured in conventional static two-dimensional conditions are physiologically not representative due to their lack of shear-stress and their inherent pro-inflammatory state. Novel culturing platforms that allow endothelial cell culture under these physiological conditions are needed to unravel organ-specific vascular host responses.

Methods

Here, we present novel organ-specific three-dimensional primary endothelial vessel-on-chip microfluidic models for studying orthohantavirus infection. We established a high-throughput method for culturing these vessels originating from lungs, kidney, liver, heart, skin and umbilical vein.

Results

Upon TNFα-stimulation, endothelial cell activation leads to increases of inflammation marker ICAM-1 expression, monocyte adhesion, platelet binding and vascular permeability. Furthermore, we demonstrated that Puumala orthohantavirus infection causes endothelial cell activation in primary human umbilical vein endothelial cells resulting in increased monocyte adhesion without directly causing vascular permeability increases.

Conclusions

These results characterize a novel three-dimensional vessel-on-chip platform to study orthohantavirus pathogenesis. This platform can be expanded by addition of co-cultures with other (immune) cell types to optimize representation of organ-specific microenvironments. Ultimately, this platform will pave the way for studying pathogenesis or assessment of possible therapeutics for a wide range of endotheliotropic viruses.

Background and Aims

SARS-CoV-2 has infected hundreds of millions globally and resulted in nearly 6 million deaths to date, with ongoing waves of infection in communities with relatively high vaccination rates. We still face many challenges in the rollout of vaccines and subsequent boosting of initial protective immune responses. A high-density microarray patch (HD-MAP) has been developed at the University of Queensland and Vaxxas that can deliver vaccines to the skin, with significant dose sparing and high thermostability.

Methods

This presentation will cover the background HD-MAP technology and its application to COVID-19 vaccine development. We have used the HD-MAP to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin of mice in order to examine protective efficacy.

Results

We have shown that the vaccine, dry-coated on the patch is thermostable, and delivery of spike via HD-MAP induces higher cellular and antibody immune responses than traditional needle delivery, with serum able to potently neutralize clinically relevant isolates including alpha, beta, delta and omicron lineages. Finally, a single dose of HD-MAP-delivered spike provided complete protection from a lethal virus challenge.

Conclusions

We have shown that HD-MAP delivery of a SARS-CoV-2 vaccine has the potential to significantly impact the ongoing COVID-19 pandemic.

Background and Aims

Chronic wounds can readily become infected by microorganisms due to failure of mechanical and physiological first line innate immune responses. We report the characterization of host adaptation of three Escherichia coli ST131 genomes that occurred during a 10-year chronic wound infection after a foot fracture.

Methods

The three E. coli strains were characterized by various microbiological and genomic approaches as assessment of antimicrobial resistance, growth in different media, biofilm formation and genome sequencing by PacBio RSII. Phylogenetic analyses and genome alterations such as single nucleotide polymorphisms, deletion and rearrangements that led to pseudogenes and chromosomal inversions were documented. Relevant selected metabolic and physiological pathways were analyzed for integrity.

Results

The three E. coli ST131 strains showed a heavily host-adapted genome with high number of pseudogenes and a large chromosomal inversion compared to ST131 reference strains. Furthermore, two of three E. coli ST131 isolates were small colony variants with its genetic basis in multiple genome alterations including pseudogenes and deletions in the pathway for heme biosynthesis. Pseudogene analysis indicated three ST131 strains to be mutator strains. Enhanced capability of biofilm formation of the ST131 isolates was indicated by the agar plate assay.

Conclusions

ST131 clone members are ubiquitously found in patients and the environment. ST131 strains have perhaps already been acquired from the environment upon foot fracture and persisted in wounds showing outmost genome plasticity and adaptability which might causing the chronic infection. Although co-infection with E. faecalis might have supported chronicity, these findings indicate that in individuals with underlying metabolic diseases wound infection by ST131 E. coli isolates can be a health risk.

Background and Aims

Burkholderia cenocepacia is a Gram-negative opportunistic bacterium known to cause severe lung infections in people with cystic fibrosis. Two main extracellular proteases, being zinc metalloproteases ZmpA and ZmpB, potentially shape the degree of pathogenicity^(1,2). Additionally, a BCAM1744 orthologue of serine metalloprotease PrtA (solely responsible for extracellular proteolytic activity of B. glumae⁽³⁾) is highly expressed in B. cenocepacia. However, the exact involvement of these three extracellular proteases during infection remains to be elucidated.

Methods

To understand this, we heterologously produced these proteases in E. coli and studied their impact on several substrates through mass spectrometry. Besides analyzing specific molecules mimicking in vivo target(s), the impact on several synthetic peptides was also investigated to perceive their substrate specificity. Parallel, B. cenocepaciaK56-2 knock-out mutants were constructed which were subsequently analyzed in terms of their fitness and extracellular proteolytic activities.

Results

Intriguingly, our data demonstrated high degree of similarity in their substrate specificity, as well as their ability to compensate each other’s absence. This thus shows that ZmpA, ZmpB and BCAM1744 might execute the same tasks in a synchronized fashion at a different stage of infection.

Conclusions

Results reinforce the genomic versatility of B. cenocepaciato express different weaponry/extracellularly means for niche-adaptation. Affirmative understanding of such pathogenic behavior will eventually unravel the intracellular survival machinery resolving novel antimicrobial targets.

Acknowledgment:

This work was supported by a grant from the FWO-Vlaanderen (3G005719).

1. Gingues, S. (2005) J Bacteriol 187(13):4421–9.

2. Kooi, C. (2006) Infect Immunit 74(7):4083–93.

3. Lelis, T. (2019) Mol Plant-Microbe Interact 31(7):841–52.