Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

Myosin IIA is essential for Shigella flexneri cell-to-cell spread.

A key feature of Shigella pathogenesis is the ability to spread from cell-to-cell post-invasion. This is dependent on the bacteria's ability to initiate de novo F-actin tail polymerisation, followed by protrusion formation, uptake of bacteria-containing protrusion and finally, lysis of the double membrane vacuole in the adjacent cell. In epithelial cells, cytoskeletal tension is maintained by the actin-myosin II networks. In this study, the role of myosin II and its specific kinase, myosin light chain kinase (MLCK), during Shigella intercellular spreading was investigated in HeLa cells. Inhibition of MLCK and myosin II, as well as myosin IIA knockdown, significantly reduced Shigella plaque and infectious focus formation. Protrusion formation and intracellular bacterial growth was not affected. Low levels of myosin II were localised to the Shigella F-actin tail. HeLa cells were also infected with Shigella strains defective in cell-to-cell spreading. Unexpectedly loss of myosin IIA labelling was observed in HeLa cells infected with these mutant strains. This phenomenon was not observed with WT Shigella or with the less abundant myosin IIB isoform, suggesting a critical role for myosin IIA.

Dynamin-related protein Drp1 and mitochondria are important for Shigella flexneri infection.

Shigella infection in epithelial cells induces cell death which is accompanied by mitochondrial dysfunction. In this study the role of the mitochondrial fission protein, Drp1 during Shigella infection in HeLa cells was examined. Significant lactate dehydrogenase (LDH) release was detected in the culture supernatant when HeLa cells were infected with Shigella at a high multiplicity of infection. Drp1 inhibition with Mdivi-1 and siRNA knockdown significantly reduced LDH release. HeLa cell death was also accompanied by mitochondrial fragmentation. Tubular mitochondrial networks were partially restored when Drp1 was depleted with either siRNA or inhibited with Mdivi-1. Surprisingly either Mdivi-1 treatment or Drp1 siRNA-depletion of HeLa cells also reduced Shigella plaque formation. The effect of Mdivi-1 on Shigella infection was assessed using the murine Sereny model, however it had no impact on ocular inflammation. Overall our results suggest that Drp1 and the mitochondria play important roles during Shigella infection.

Impact of dynasore an inhibitor of dynamin II on Shigella flexneri infection.

Shigella flexneri remains a significant human pathogen due to high morbidity among children < 5 years in developing countries. One of the key features of Shigella infection is the ability of the bacterium to initiate actin tail polymerisation to disseminate into neighbouring cells. Dynamin II is associated with the old pole of the bacteria that is associated with F-actin tail formation. Dynamin II inhibition with dynasore as well as siRNA knockdown significantly reduced Shigella cell to cell spreading in vitro. The ocular mouse Sereny model was used to determine if dynasore could delay the progression of Shigella infection in vivo. While dynasore did not reduce ocular inflammation, it did provide significant protection against weight loss. Therefore dynasore's effects in vivo are unlikely to be related to the inhibition of cell spreading observed in vitro. We found that dynasore decreased S. flexneri-induced HeLa cell death in vitro which may explain the protective effect observed in vivo. These results suggest the administration of dynasore or a similar compound during Shigella infection could be a potential intervention strategy to alleviate disease symptoms.

IcsA autotransporter passenger promotes increased fusion protein expression on the cell surface.

BACKGROUND: Autotransporters are attractive cell surface display vehicles as they lack complex adaptor proteins necessary for protein export. Recent reports have suggested that the native effector domain (α domain) and translocation domain (ß domain) interact with each other to drive translocation of the effector domain to the outer membrane. In this report we compared the expression, surface localisation and folding of TEM-1 ß-lactamase (Bla) and maltose binding protein (MalE or MBP) fused to either full length Shigella flexneri IcsA (IcsA) autotransporter or to the ß domain alone (IcsAß) to determine the contribution of the native IcsA α domain in presenting the fusion proteins on the surface of E. coli K-12 UT5600 (ΔompT). RESULTS: Expression of IcsA-Bla was greater than IcsAß-Bla. High levels of IcsA-MalE were detected but IcsAß-MalE was not expressed. All fusion proteins other than IcsAß-MalE were localised to the outer membrane and were detected on the surface of UT5600 via immunofluorescence microscopy. All bacteria expressing IcsA-MalE were labelled with both α-IcsA and α-MBP. UT5600 expressing IcsAß-MalE was not labelled with α-MBP. A third of UT5600 expressing IcsA-Bla were detectable with α-Bla but only 5% of UT5600 (IcsAß-Bla) were labelled with α-Bla. The correct folding of the Bla moiety when fused to IcsA and IcsAß was also retained as UT5600 expressing either fusion protein exhibited a decreased zone of inhibition in the presence of ampicillin. UT5600 expressing IcsA-Bla was more resistant compared to UT5600 expressing IcsAß-Bla. CONCLUSIONS: The export mechanism of autotransporters is not well understood but accumulating evidence suggest a critical role for the native effector or α domain in facilitating its own export via interactions with the translocation or ß domain. This is the first report directly comparing expression of heterologous proteins fused to the full length IcsA autotransporter and fusion to the ß domain alone. Protein expression and surface presentation of the fusion proteins were dramatically improved when fused to IcsA rather than IcsAß. Future studies involved in designing autotransporters as cell surface display vehicles would benefit from including the native α domain. This work also provides further evidence for a key interaction between the autotransporter α and ß domains.