Biology subjects

Naesens, L. M. J.

Publications and source records attributed to Naesens, L. M. J..

3 recordsLinked to original sources

bioRxiv2025

Random mutagenesis of influenza hemagglutinin identifies new sites which modulate its acid-stability and cleavability

The structural instability of influenza hemagglutinin (HA) is related to its function in low pH-mediated membrane fusion, which requires prior cleavage of the premature HA0 by a host protease. The precise determinants underlying the stability and cleavability of HA remain to be fully understood and have implications for risk assessment of zoonotic influenza A viruses (IAV), viral transmissibility and vaccine production. To address this, we conducted random mutagenesis on early 2009 pandemic H1 HA, followed by selection of acid-stable viruses and detailed profiling of the mutant HAs. This resulted in identification of four mutations, which increase the acid-stability and decrease the fusion-promoting activity of H1 HA, without compromising viral entry and replication in cells. The newly recognized mutations are situated in the globular head, vestigial esterase and membrane-proximal part of H1 HA, in regions involved in the refolding of HA at low pH. A fifth mutation, D346N, is located in the cleavage loop and renders H1 HA0 12-fold resistant to trypsin activation, whereas its cleavage by transmembrane serine protease 2 (TMPRSS2) is not affected. Along this line, we found that the poor cleavage of H16 HA0, which is unusual in carrying an N346 residue, only applies when it is performed by extracellular proteases. Since H16 HA also exhibits a very low fusion pH, we propose that gull H16N3 virus may carry a much more stable HA than other avian IAVs. Collectively, our mutagenesis approach revealed new determinants of HA stability and cleavability, with relevance for viral surveillance and vaccine production. IMPORTANCEThe presence of influenza A viruses (IAV) throughout the animal world, particularly avian species, represents a constant threat for zoonotic infections or a new influenza pandemic. To be transmissible among humans, a zoonotic IAV requires mutations in the viral hemagglutinin (HA) that increase the acid-stability of this mediator of viral entry. Understanding the determinants of HA stability is also important to produce vaccines with high shelf-stability. By combining random mutagenesis with selection of acid-stable viruses, we identified new stabilizing mutations located in different parts of HA. Besides, we discovered a mutation that renders HA resistant to cleavage by extracellular proteases. Since this residue is naturally occurring in H16 HA, we propose that the gull H16N3 virus may differ from other avian IAVs in carrying an environmentally stable HA. Hence, our study delivers new insight in factors that modulate HA acid-stability and cleavability, with relevance for viral surveillance and vaccine production.

microbiology↗

bioRxiv2024

Novel Pentafluorosulfanyl-containing Triclocarban Analogs selectively kill Gram-positive bacteria.

The antibacterial and antibiofilm efficacy of our novel pentafluorosulfanyl-containing triclocarban analogs was explored against seven different Gram-positive and Gram-negative indicator strains. After initial screening, they had bactericidal and bacteriostatic activity against Gram-positive bacteria, especially Staphylococcus aureus and MRSA (methicillin-resistant staphylococcus aureus) in a very low concentration. Our results were compared with the most common antibiotic being used (Ciprofloxacin and Gentamycin); the novel components had significantly better antibacterial and antibiofilm activity in lower concentrations in comparison to the antibiotics. For instance, EBP-59 minimum inhibitory concentration was < 0.0003 mM, while ciprofloxacin 0.08 mM. Further antibacterial activity of novel components was surveyed against 10 clinical antibiotic resistance MRSA isolates. Again, novel components had significantly better antibacterial and antibiofilm activity in comparison with antibiotics. Mechanistic studies have revealed that none of these novel compounds exhibit any effect on the reduced thiol, disrupting iron sulfur clusters, or hydrogen peroxide pathways. Instead, their impact is attributed to the disruption of the Gram-positive bacterial cell membrane. Toxicity and safety testing on tissue cell culture showed promising results for the safety of components to the host.

microbiology↗

bioRxiv2022

Effect of SARS-CoV-2 spike mutations on its activation by TMPRSS2 and TMPRSS13

The continuous emergence of new SARS-CoV-2 variants urges better understanding of the functional motifs in the spike (S) protein and their tolerance towards mutations. We here focus on the S2 motif which, during virus entry, requires cleavage by a cell surface protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2 motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant S-pseudotyped viruses, we show that K814, which precedes the scissile R815 residue, is dispensable for SARS-CoV-2 spike activation by TMPRSS2 but not TMPRSS13. The latter protease lost its activity towards SARS-CoV-2 S when the S2 motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689) and also the reverse effect was seen. This swap had no impact on TMPRSS2 activation. Also in the MERS-CoV spike, introducing a dibasic scissile motif was fully accepted by TMPRSS13 but less so by TMPRSS2. Our findings are the first to demonstrate which S2 residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS13 exhibiting higher preference for K/R rich motifs than TMPRSS2. This preemptive insight can help to estimate the impact of S2 motif changes as they may appear in new SARS-CoV-2 variants. IMPORTANCESince the start of the COVID-19 pandemic, SARS-CoV-2 is undergoing worldwide selection with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning in a direct manner. Remarkably, though located in a strong immunogenic region, the S2 cleavage motif has, thus far, remained highly conserved. This suggests that its amino acid sequence is critical for spike activation by airway proteases. To investigate this, we assessed which S2 site mutations affect processing by TMPRSS2 and TMPRSS13, two main activators of the SARS-CoV-2 spike. Being the first in its kind, our study will help to assess the biological impact of S2 site variations as soon as they are detected during variant surveillance.

microbiology↗