Biology subjects

Silva-Bea, S.

Publications and source records attributed to Silva-Bea, S..

3 recordsLinked to original sources

bioRxiv2026

SdiA as a Repressor of Phagocytosis and Intracellular Survival in Klebsiella pneumoniae: Insights from Macrophage and Amoeba Models

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI

microbiology↗

bioRxiv2024

Lineage-specific neutralising antibodies after SARS-CoV-2 mild disease. Immune boosting effect of vaccination

We followed a group of 105 non-vaccinated individuals after Alpha or Delta SARS-CoV-2 mild disease, measuring the viral shedding (qRT-PCR, dPCR and subgenomic RNA-E) and humoral response (commercial immunoassay and pseudovirus and live virus neutralisation) up to six months. Sixty nine patients received a vaccination boost during the follow-up period (n=95). Subgenomic RNA-E showed a shorter period until negativity (mean 2.2 weeks) compared to gRNA (mean 5.2 weeks). A high correlation between qRT-PCR and dPCR was found for viral load estimation, even when no nucleic acid extraction was used in dPCR (R2 = 0.87). Post-convalescent sera showed the strongest neutralisation against the variant of natural exposure, while the neutralisation capacity against Omicron variants was significantly lower compared to the other variants. Additionally, the results suggested that commercial immunoassays may not accurately predict protection against a different variant than the variant of exposure. An immune boosting effect of the SARS-CoV-2 vaccination was evident. Variant-specific neutralising antibodies were detected one month after natural infection. Although short lived, maximum igG response was observed after hybrid immunisation (natural infection + vaccination). This study also points to potential improvements in the clinical management of SARS-CoV-2 cases. Firstly, subgenomic RNA-E is a potentially more accurate biomarker of infectivity than current qRT-PCRs using genomic RNA as target. Secondly, accuracy of high-throughput immunoassays must be validated in order to estimate specific protection and organise vaccination campaigns. Our findings could play a role in the current implementation of SARS-CoV-2 vaccine programs. Author SummaryYears after SARS-CoV-2 related infections challenged the healthcare systems of the whole world, the optimal strategy to deal with diagnosis, quarantines or vaccination patterns is still a matter of debate. The interplay between infectivity and immunity in the different circulating variants is complex, and qPCR for diagnosis may extend quarantines, as detection of viral RNA does not necessarily mean that the virus remains infectious. We studied a group of patients infected with pre-Omicron variants to study how their variant-specific antibodies reacted to the past and present variants of SARS-CoV-2. In both Alpha and Delta, antibodies neutralise their own variants better than other variants that came before and after. Perhaps most importantly, neutralisation was lowest against Omicron variants. We followed the viral shedding dynamics of the patients, testing different PCR techniques and targets. Subgenomic RNA was detectable in nasopharyngeal samples for a shorter time than genomic RNA and it has been suggested as a good marker of infectivity. Using sgRNA instead of genomic RNA as a PCR target could reduce hospital bed occupation and quarantine time. Overall, we hope that these results could help guide pandemic and diagnostic control in the future.

microbiology↗

bioRxiv2024

Regulatory effects on virulence and phage susceptibility revealed by sdiA mutation in Klebsiella pneumoniae

The World Health Organization has identified multi-drug resistant (MDR) Klebsiella pneumoniae strains as the highest priority in 2024. SdiA, a LuxR-like quorum sensing (QS) receptor that responds to N-acyl-homoserine lactones (AHLs), exerts a substantial regulatory influence on the virulence of numerous Gram-negative bacteria. The function of this receptor in the virulence of K. pneumoniae remains uncertain. Nevertheless, further investigation into the significance of this receptor is needed, as it represents an intriguing avenue with the potential to contribute to the development of novel antimicrobial strategies. The objective of the present study was to elucidate the function of SdiA in K. pneumoniae biofilm formation and virulence. To this end, a genetic knockout of sdiA was conducted, and virulence-related phenotypic studies were performed following AHL provision. The results demonstrate that SdiA deficiency increases susceptibility to phage infection and human serum resistance, and promotes biofilm maturation and cell filamentation. No effect on virulence was observed in vivo in the Galleria mellonella infection model. The addition of N-hexanoyl-L-homoserine lactone (C6-HSL) promoted SdiA-dependent biofilm maturation but also enhanced serum resistance and reduced virulence against G. mellonella in the absence of SdiA. The results of this study demonstrate that C6-HSL and SdiA exert a dual influence on virulence phenotypes, operating both independently and hierarchically. These findings provide new insights into the virulence of K. pneumoniae and its regulation by SdiA. ImportanceThis study represents a significant contribution to our understanding of the complex regulatory mechanisms that govern the virulence of multi-drug resistant Klebsiella pneumoniae through quorum sensing (QS). The study offers insights into the function of SdiA, a QS receptor, in the regulation of biofilm formation, susceptibility to phage infection, serum resistance, and cell filamentation in this bacterium. Furthermore, the findings of this study demonstrate that exogenous N-acyl-homoserine lactone (AHL) signalling influences the aforementioned virulence phenotypes in both a SdiA-dependent and independent manner, as well as in a hierarchical manner.

microbiology↗