Welcome to the IUMS 2022 Interactive Program

Abstract Body

Cholera is a devastating diarrheal disease that sickens millions of people each year. Despite incredible progress over the past hundred years in our understanding of the pathogen’s virulence mechanisms, the environmental aspects of the causative agent of the disease, the bacterium Vibrio cholerae, have so far been insufficiently studied at the molecular level. In my talk, I will address this knowledge gap and present insights into the pathogen’s environmental lifestyle including its potential for interbacterial competition on biotic surfaces and its evolvability. I will also show how the bacterium defends itself against mobile genetic elements such as plasmids and phages, which involves dedicated DNA defense systems that are unique to the seventh pandemic clade of this cholera bcteria. I will end my talk with speculations on how these features might have shaped the evolution of the most successful lineage of pandemic V. cholerae.

Abstract Body

Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. When infecting the host, M. tuberculosis is internalized by immune cells, primarily macrophages. One of the mechanisms by which M. tuberculosis is able to survive and proliferate within these infected cells is by permeating the phagosomal membrane using secreted effector proteins. Also, other secreted mycobacterial virulence factors manipulate the host cell defenses and allow the bacterium to adapt to the hostile macrophage environment.

In order to secrete virulence proteins across the cell envelope, M. tuberculosis and also other pathogenic mycobacteria face the same problem as Gram-negative bacteria, they have to secret proteins across a diderm cell envelope. In mycobacteria this is achieved by a specialized secretion system known as the type VII secretion system (T7S), which is also named the ESX secretion system. The genome of M. tuberculosis encodes five of these ESX secretion systems and it has been estimated that more than hundred substrates are transported to the cell surface or in the environment. In our research we focus on the substrates of the ESX-1 and ESX-5 systems and the role they play in the interaction with the host environment and we sue the fish pathogen M. marinum asmodel organism. The ESX-1 secretion system is required for full pathogenicity and is responsible for the secretion of a set of crucial virulence factors that play a key role in the escape of mycobacteria from the phagosome to the cytosol during infection. ESX-5 is an essential secretion system required for nutrient uptake and membrane permeability. Moreover, it secretes a vast number of substrates, including most PE and PPE proteins, which play a role in the host immunomodulation and immune evasion. One of the type VII substrates is CpnT, the only toxin of M. tuberculosis that has been identified thus far. The activity of multiple ESX systems seems to be required to secret this toxin in host macrophages.

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

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.

Background and Aims

To control COVID-19, elucidation of SARS-CoV-2 replication mechanisms is required. In this study, we tried to identify the host factors for SARS-CoV-2 infection in human cells through the human whole-genome screening using the CRISPR/Cas9 system.

Methods

A549-hACE2 cells expressing Cas9 were transfected with a gRNA library using lentiviral vector. The cells were inoculated with purified SARS-CoV-2/UT-NCGM02/Human/2020/Tokyo at MOI of 0.3. Three days after inoculation, DNA was extracted from the cells and applied to next-generation sequencing analysis.

Results

We identified TRIM28 and TRIM33 as new candidate genes. We suppressed the expressions of the genes in cultured cells by using CRISPRi to confirm their involvement in the viral infection. TRIM28 and TRIM33 knockdown reduced viral titer by 2.0 log and more than 5.0 log, respectively. When viral RNA levels in infected cells and supernatant were quantified, TRIM33 knockdown cells showed a decrease in viral RNA levels in both infected cells and supernatant, while TRIM28 showed an increase in RNA levels in both. Consistent with this, viral nucleocapsid protein expression in infected cells was decreased in TRIM33 knockdown and increased in TRIM28 knockdown. SARS-CoV-2 performs particle assembly and budding at the ER Golgi intermediate (ERGIC), however, in the TRIM28 knockdown cells, retention of viral Spike protein in the ERGIC was not observed.

Conclusions

These findings suggest that in the SARS-CoV-2 replication cycle, TRIM28 is involved in the assembly of infectious particles and TRIM33 is engaged in the cell entry and/or transcriptional replication.

Background and Aims

Hepatitis B virus (HBV) affects about 250 million individuals globally and is a major cause of chronic liver inflammation. We aimed to identify the host factors that support HBV/HDV infection and to target them as a new therapeutic modality.

Methods

We performed functional siRNA screening using an HBV reporter virus and HepG2-hNTCP cells to uncover host factors that impact the HBV life cycle.

Results

We found that The Kinesin KIF4 facilitates HBV, and iHDV, entry into human hepatocytes. Cellular fractionation and immunofluorescence analysis (IF) showed that transient KIF4 depletion reduced surface and raised intracellular NTCP levels leading to the suppression of both HBV and HDV infection. Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored cell permissiveness to HBV. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study showed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and suppression of subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC₅₀ 1.89 ± 0.98 μM) cultures.

Conclusions

Our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry