Roach, E. J. | bioRxiv Science

bioRxiv2021

Dual perspective proteomics infectome profiling discovers Salmonella type III secretion system effector functions in macrophages

Intracellular bacterial pathogens have evolved sophisticated infection strategies, including the release and secretion of virulence factors to interfere with host cell functions and to perturb immune responses. For Salmonella enterica serovar Typhimurium (S. Typhimurium), the type III secretion systems encoded on Salmonella pathogenicity islands (SPI) 1 and 2 mediates invasion of the bacterium into innate immune cells and regulates bacterial replication and survival within the hostile environment of the host, respectively. Here, we explore the temporal and strain-specific dual perspective response of both the host and pathogen during cellular infection via quantitative proteomics. We report time- and pathogenicity island-specific expression and secretion of infection-associated proteins (i.e., SL1344_1263, SL1344_3112, SL1344_1563, and YnhG) and regulated immune response proteins in macrophage, including Cd86, Cd40, Casp4, C3, IL-1, and Cd69). Through intracellular macrophage and in vivo murine models of infection, we reveal a role in virulence for three of the bacterial proteins (SL1344_1263, SL1344_1563, and YnhG), defining their importance as novel T3SS effectors. We characterize the temporal intra- and extracellular production of the effectors and identify their interaction networks in host cells, representing inhibitory and stimulatory pathways mounted by invading bacterial pathogens. Author SummaryThe relationship between a host and pathogen is intricate, and regulation of the host immune response correlates with the progressive timing of infection and tailored responses to the pathogen. Relying on detection and quantification of protein-level changes using mass spectrometry-based proteomics, we explore the production of known and novel effectors by Salmonella during intracellular survival within macrophage. Our results portray a role for these effectors in bacterial virulence using an in vivo murine model of infection, and we define a dynamic network of interaction between the effectors and host proteins. These interactions reveal opportunity for drug design to disrupt modulation of the host by the invading bacterium as a new strategy for combatting infection. Our approach is dynamic and universal, with the power to alter therapeutic discovery against infectious diseases.

microbiology↗

bioRxiv2019

Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems (https://doi.org/10.1101/769745). Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3,5-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of Bacilli, Clostridia, Streptococci, and Actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.\n\nAuthor summaryThe Pel polysaccharide is required for biofilm formation in P. aeruginosa and we have previously found that the genes necessary for biosynthesis of this polymer are broadly distributed across Gram-negative bacteria. Herein, we show that many species of Gram-positive bacteria also possess Pel biosynthetic genes and demonstrate that these genes are used Bacillus cereus for biofilm formation. We show that Pel production in B. cereus is regulated by c-di-GMP and have identified two enzymes, a diguanylate cyclase, CdgF, and a phosphodiesterase, CdgE, that control the levels of this bacterial signalling molecule. While Pel production in B. cereus also requires the binding of c-di-GMP to the receptor PelD, the divergence of this protein in Streptococci suggests a c-di-GMP independent mechanism of regulation is used in this species. The discovery of a Pel biosynthetic gene cluster in Gram-positive bacteria and our characterization of the pel operon in B. cereus suggests that Pel is a widespread biofilm component across all bacteria.

microbiology↗