Biology subjects

Sa-Pessoa, J.

Publications and source records attributed to Sa-Pessoa, J..

4 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↗

bioRxiv2022

Modelling the gastrointestinal carriage of Klebsiella pneumoniae infections.

Klebsiella pneumoniae is a leading cause of nosocomial and community acquired infections, making K. pneumoniae the second pathogen associated with the most deaths attributed to any antibiotic resistant infection. K. pneumoniae colonises the nasopharynx and the gastrointestinal tract in an asymptomatic manner without dissemination to other tissues; importantly gastrointestinal colonisation is a requisite for infection. Our understanding of K. pneumoniae colonisation is still based on interrogating mouse models in which animals are pre-treated with antibiotics to disturb the colonisation resistance imposed by the gut microbiome. In these models, infection disseminates to other tissues. Here, we report a murine model to allow for the study of the gastrointestinal colonisation of K. pneumoniae without tissue dissemination. Hypervirulent and antibiotic resistant strains stably colonise the gastrointestinal tract of in an inbred mouse population without antibiotic treatment. The small intestine is the primary site of colonisation followed by a transition to the colon over time without dissemination to other tissues. Our model also mimics the disease dynamics of metastatic K. pneumoniae strains able to disseminate from the gastrointestinal tract to other sterile sites. Colonisation is associated with mild to moderate histopathology, no significant inflammation, and no effect on the richness of the microbiome. Our model recapitulates the clinical scenario in which antibiotic treatment disturbs the colonisation of K. pneumoniae resulting in dissemination to other tissues. Finally, we establish that the capsule polysaccharide is necessary for the colonisation of the large intestine whereas the type VI secretion system contributes to colonisation across the gastrointestinal tract. IMPORTANCEKlebsiella pneumoniae is one of the pathogens sweeping the World in the antibiotic resistance pandemic. Klebsiella colonises the nasopharynx and the gut of healthy subjects in an asymptomatic manner, being gut colonisation a requisite for infection. This makes essential to understand the gastrointestinal carriage to prevent Klebsiella infections. Current research models rely on the perturbation of the gut microbiome by antibiotics, resulting in an invasive infection. Here, we report a new model of K. pneumoniae gut colonisation that recapitulates key features of the asymptomatic human gastrointestinal tract colonisation. In our model, there is no need to disturb the microbiota to achieve stable colonization without dissemination to other tissues. Our model recapitulates the clinical scenario in which antibiotic treatment triggers invasive infection. We envision our model will be an excellent platform to test therapeutics to eliminate Klebsiella asymptomatic colonisation, and to investigate factors enhancing colonisation and invasive infections.

microbiology↗

bioRxiv2020

Klebsiella pneumoniae reduces SUMOylation to limit host defence responses

Klebsiella pneumoniae is an important cause of multidrug resistant infections worldwide. Understanding the virulence mechanisms of K. pneumoniae is a priority and timely to design new therapeutics. Here we demonstrate that K. pneumoniae limits the SUMOylation of host proteins in epithelial cells and macrophages (mouse and human) to subvert cell innate immunity. Mechanistically, in lung epithelial cells Klebsiella increases the levels of the deSUMOylase SENP2 in the cytosol by affecting its K48-ubiquitylation and its subsequent degradation by the ubiquitin proteasome. This is dependent on Klebsiella preventing the NEDDylation of the Cullin-1 subunit of the ubiquitin ligase complex E3-SCF{beta}-TrCP by exploiting the CSN5 deNEDDylase. Klebsiella induces the expression of CSN5 in an EGFR-PI3K-AKT-ERK-GSK3{beta} signalling pathway dependent manner. In macrophages, TLR4-TRAM-TRIF induced type-I IFN via IFNAR1-controlled signalling mediates Klebsiella-triggered decrease in the levels of SUMOylation via let-7 miRNAs. Our results revealed the crucial role played by Klebsiella polysaccharides, the capsule and the LPS O-polysaccharide, to decrease the levels of SUMO-conjugated proteins in epithelial cells and macrophages. Klebsiella-induced decrease in SUMOylation promotes infection by limiting the activation of inflammatory responses and increasing intracellular survival in macrophages. IMPORTANCEKlebsiella pneumoniae has been singled out as an urgent threat to human health due to the increasing isolation of strains resistant to "last line" antimicrobials, narrowing the treatment options against Klebsiella infections. Unfortunately, at present, we cannot identify candidate compounds in late-stage development for treatment of multidrug Klebsiella infections; this pathogen is exemplary of the mismatch between unmet medical needs and the current antimicrobial research and development pipeline. Furthermore, there is still limited evidence on K. pneumoniae pathogenesis at the molecular and cellular level in the context of the interactions between bacterial pathogens and their hosts. In this research, we have uncovered a sophisticated strategy employed by Klebsiella to subvert the activation of immune defences by controlling the modification of proteins. Our research may open opportunities to develop new therapeutics based on counteracting this Klebsiella-controlled immune evasion strategy.

microbiology↗

bioRxiv2019

A porcine ex vivo lung perfusion model to investigate bacterial pathogenesis

The use of animal infection models is essential to understand microbial pathogenesis and to develop and test treatments. Insects, and 2D and 3D tissue models are increasingly being used as surrogate for mammalian models. However, there are concerns whether these models recapitulate the complexity of host-pathogen interactions. Here, we developed the ex vivo lung perfusion (EVLP) model of infection using porcine lungs to investigate Klebsiella pneumoniae-triggered pneumonia as model of respiratory infections. The porcine EVLP model recapitulates features of K. pneumoniae-induced pneumonia lung injury. This model is also useful to assess the pathogenic potential of K. pneumoniae as we observed that the attenuated Klebsiella capsule mutant strain caused less pathological tissue damage with a concomitant decrease in the bacterial burden compare to lungs infected with the wild type. The porcine EVLP model allows assessment of inflammatory responses following infection; similar to the mouse pneumonia model, we observed an increase of il-10 in the lungs infected with the wild type and an increase of ifn-{gamma} in lungs infected with the capsule mutant. This model also allows monitoring phenotypes at the single-cell level. Wild-type K. pneumoniae skews macrophages towards an M2-like state. In vitro experiments probing pig bone marrow-derived macrophages uncovered the role of the M2 transcriptional factor STAT6, and that Klebsiella-induced il10 expression is controlled by p38 and ERK. Klebsiella-induced macrophage polarization is dependent on the capsule. Altogether, this study support the utility of the EVLP model using pig lungs as platform to investigate the infection biology of respiratory pathogens.\n\nIMPORTANCEThe implementation of infection models that approximate human disease is essential to understand infections and for testing new therapies before they enter into clinical stages. Rodents are used in most of pre-clinical studies, although the differences between mouse and man have fuelled the conclusion that murine studies are unreliable predictors of human outcomes. Here, we have developed a whole lung porcine model of infection using the established ex vivo lung perfusion (EVLP) system established to re-condition human lungs for transplant. As a proof-of-principle, we provide evidence demonstrating that infection of the porcine EVLP with the human pathogen K. pneumoniae recapitulates the known features of Klebsiella-triggered pneumonia. Moreover, our data revealed the porcine EVLP model is useful to reveal features of the virulence of K. pneumoniae including the manipulation of immune cells. Altogether, this study supports the utility of the EVLP model using pig lungs as surrogate host for assessing respiratory infections.

microbiology↗