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The serum complement system is a first line of defense against bacterial invaders. Resistance to killing by serum enhances the capacity of Klebsiella pneumoniae to cause infection, but it is an incompletely understood virulence trait. Identifying and characterizing the factors responsible for preventing activation of, and killing by, serum complement could inform new approaches to treatment of K. pneumoniae infections. Here, we used functional genomic profiling to define the genetic basis of complement resistance in four diverse serum-resistant K. pneumoniae strains (NTUH-K2044, B5055, ATCC 43816, and RH201207), and explored their recognition by key complement components. More than 90 genes contributed to resistance in one or more strains, but only three, rfaH, lpp, and arnD, were common to all four strains. Deletion of the antiterminator rfaH, which controls the expression of capsule and O side chains, resulted in dramatic complement resistance reductions in all strains. The murein lipoprotein gene lpp promoted capsule retention through a mechanism dependent on its C-terminal lysine residue; its deletion led to modest reductions in complement resistance. Binding experiments with the complement components C3b and C5b-9 showed that the underlying mechanism of evasion varied in the four strains: B5055 and NTUH-K2044 appeared to bypass recognition by complement entirely, while ATCC 43816 and RH201207 were able to resist killing despite being associated with substantial levels of C5b-9. All rfaH and lpp mutants bound C3b and C5b-9 in large quantities. Our findings show that, even among this small selection of isolates, K. pneumoniae adopts differing mechanisms and utilizes distinct gene sets to avoid complement attack.

In vitro serum deprivation (SD) is one model for investigating the molecular mechanisms underlying apoptosis as well as autophagy, which generally function as defense strategies upon cell injury by eliminating damaged organelles [1]. Furthermore, SD injury in vitro is widely used to mimic the ischemic environment [2]. In serum deprived conditions, cells show different parameters of apoptosis and autophagy. Melatonin (MLT), a lipophilic indole secreted by pineal and non-pineal cells, is a well-known potent free radical scavenger acting as neuroprotective molecule that prevents apoptotic cell death in several models of neurodegenerative diseases. In the present study we investigated the neuroprotective effects of MLT during SD condition on mouse hippocampal HT22 cells, considering that intracellular ROS are usually linked to autophagy and apoptosis. To explore potential effects of combining SD with melatonin we studied clonogenic survival of HT22 cells. Clonogenic assay demonstrated a significative (p< 0.01) reduction of HT22 total cell numbers challenged for 24h with SD, whereas the pre-treatment with 200nM of MLT for 24hr noticeably reduced this effect of about 30%. In HT22 starved cells the percentage of MitoTracker Red (MTR) positive cells doubled (P< 0.05) if compared to the control condition, suggesting that SD induced a remodelling of mitochondrial network. It is noteworthy that MLT pre-treatment produced a MTR positivity similar to that of controls. We next investigated whether melatonin was able to influence the autophagic pathway. Autophagy was detected by measuring the aggregation of LC3B protein coupled to green fluorescence protein (GFP). Confocal images show that SD induced an increase in the GFP-LC3 puncta, whereas the melatonin treatment reduces these aggregations. Taken together, our results suggest that MLT treatment may play protective roles against cellular modifications induced by SD treatment in HT22 cells., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Problem Statement Campylobacter jejuni adheres, invades and resides within intestinal epithelial cells (IECs). However, the exact process at the cellular level leading to diarrhoeal disease is poorly understood. There have been studies that link intestinal inflammation and the unfolded protein response (UPR), which is a conserved pathway to restore homeostasis in the endoplasmic reticulum. Recent data has shown that C. jejuni activates UPR through IRE1α pathway. Here, we investigated the activation of the UPR through PERK, IRE1α, and ATF6 pathways by C. jejuni. Approach To investigate whether C. jejuni infection induces UPR in IECs, T84 cells were infected with C. jejuni 11168H, 81-176, and 488 wild-type strains with different exposure times. RNA and proteins were isolated from infected cells, and the induction of UPR by C. jejuni wild-type strains was observed using transcription and proteomic methods. Results The activation of all three UPR branches, PERK, IRE1α, and ATF6 was demonstrated by increased levels of transcription of chop, spliced xbp1, and atf6, respectively compared to uninfected control. The upregulation of these genes was confirmed by western blot, which showed increased amount of proteins for CHOP, phosphorylated eIF2α, and spliced XBP1 compared to the control. Conclusions The combined results demonstrate that C. jejuni induces the UPR through all three pathways. This study opens the way forward for improved understanding of the interaction between C. jejuni and IECs via UPR activation leading to intestinal inflammation. This understanding will further benefit the investigation of cellular pathways triggered by C. jejuni which can lead to diarrhoeal disease.

The serum complement system is a first line of defense against bacterial invaders. Resistance to killing by serum enhances the capacity of Klebsiella pneumoniae to cause infection, but it is an incompletely understood virulence trait. Identifying and characterizing the factors responsible for preventing activation of, and killing by, serum complement could inform new approaches to treatment of K. pneumoniae infections. Here, we used functional genomic profiling to define the genetic basis of complement resistance in four diverse serum-resistant K. pneumoniae strains (NTUH-K2044, B5055, ATCC 43816, and RH201207), and explored their recognition by key complement components. More than 90 genes contributed to resistance in one or more strains, but only three, rfaH, lpp, and arnD, were common to all four strains. Deletion of the antiterminator rfaH, which controls the expression of capsule and O side chains, resulted in dramatic complement resistance reductions in all strains. The murein lipoprotein gene lpp promoted capsule retention through a mechanism dependent on its C-terminal lysine residue; its deletion led to modest reductions in complement resistance. Binding experiments with the complement components C3b and C5b-9 showed that the underlying mechanism of evasion varied in the four strains: B5055 and NTUH-K2044 appeared to bypass recognition by complement entirely, while ATCC 43816 and RH201207 were able to resist killing despite being associated with substantial levels of C5b-9. All rfaH and lpp mutants bound C3b and C5b-9 in large quantities. Our findings show that, even among this small selection of isolates, K. pneumoniae adopts differing mechanisms and utilizes distinct gene sets to avoid complement attack.