Baker, C. P. | bioRxiv Science

bioRxiv2025

Candida albicans infection suppresses Lipopolysaccharide or Pseudomonas aeruginosa stimulated murine bone marrow derived macrophage (BMDM) responses

Candida albicans is a commensal fungus which populates most healthy individuals microbiota but can turn opportunistic in immunocompromised individuals and cause severe disease linked with high rates of mortality. With limited therapeutic options and increasing resistance to antifungals, novel treatment strategies for C. albicans infections is paramount. The exact immune response to C. albicans infections can be influenced by the surrounding microenvironment, for example, metabolic stresses or co-infection; although, knowledge on whether responses are enhanced or inhibited is lacking. Macrophages are a key immune cell in defence against C. albicans infection through phagocytic uptake and cytokine production that alerts other immune defence mechanisms. Here, we utilise a discovery screen approach using Data Independent Acquisition (DIA) based total proteomics to describe murine bone marrow derived macrophage (BMDM) response to C. albicans infection as well as in response to co-infection with gram-negative bacterial outer membrane component lipopolysaccharide (LPS) or live gram-negative bacteria Pseudomonas aeruginosa. We found C. albicans induced a surprisingly muted immune response in BMDMs as compared to LPS or P. aeruginosa. Moreover, upon co-infection with LPS or P. aeruginosa, C. albicans suppressed the BMDM proteome landscape and selectively suppressed BMDM secreted IL-6 and IL-12p40 cytokine responses to P. aeruginosa. Thus, C. albicans has significant suppressive capabilities in the host innate immune responses that could impact clinical outcomes during infection. Author SummaryHuman fungal pathogens are of increasing concern for global health due to infections in hospitalised patients, limited treatment options and increased resistance to antifungal treatments. The host inflammatory response to invasive infections like Candida albicans are important for trying to contain the pathogen. Pathogen evasion of this immune response poses a critical threat and can increase host morbidity and mortality. Macrophages are an important innate immune cell which recognises and responds to C. albicans infection in vivo. Here, we found that in vitro macrophage signalling, secreted cytokines and the total proteomic responses to C. albicans infection was much more subtle than the macrophage responses to live bacterial infection or the bacterial outer membrane component, lipopolysaccharide (LPS). Co-infection of C. albicans and bacteria suppressed selective macrophage protein expression including important inflammatory cytokines, IL-6 and IL-12p40. Thus, we describe an impressive suppressive response by C. albicans which poses a potential mechanism of enhanced immune evasion.

immunology↗

bioRxiv2023

Optimising Spectronaut search parameters to improve data quality with minimal proteome coverage reductions in DIA analyses of heterogeneous samples

Data independent acquisition has seen breakthroughs that enable comprehensive proteome profiling using short gradients. As the proteome coverage continues to increase, the quality of the data generated becomes much more relevant. Using Spectronaut, we show that the default search parameters can be easily optimised to minimise the occurrence of false positives across different samples. Using an immunological infection model system to demonstrate the impact of adjusting search settings we analysed mouse macrophages and compared their proteome to macrophages spiked with Candida albicans. This experimental system enabled the identification of false positives since Candida albicans peptides and proteins should not be present in the mouse only samples. We show that adjusting the search parameters reduced false positive identifications by 89% at the peptide and protein level, thereby considerably increasing the quality of the data. We also show that these optimised parameters incur a moderate cost, only reducing the overall number of true positive identifications across each biological replicate by <6.7% at both the peptide and protein level. We believe the value of our updated search parameters extends beyond a two-organism analysis and would be of great value to any DIA experiment analysing heterogenous populations of cell types or tissues.

biochemistry↗