Persisters represent a small subpopulation of non- or slow-growing bacterial cells that are tolerant to killing by antibiotics. Despite their prominent role in the recalcitrance of chronic infections to antibiotic therapy, the mechanism of their formation has remained elusive. We show that sorted cells of Escherichia coli with low levels of energy-generating enzymes are better able to survive antibiotic killing. Using microfluidics time-lapse microscopy and a fluorescent reporter for in vivo ATP measurements, we find that a subpopulation of cells with a low level of ATP survives killing by ampicillin. We propose that these low ATP cells are formed stochastically as a result of fluctuations in the abundance of energy-generating components. These findings point to a general “low energy” mechanism of persister formation.

Metastasis is a hallmark of malignant neuroblastoma and is the main reason for therapeutic failure and recurrence of the tumor. The CXC chemokine receptor-4 (CXCR4), a Gi protein-coupled receptor for the ligand CXCL12/stromal cell-derived factor-1α (SDF-1α), is expressed in various types of tumor. This receptor mediates the homing of tumor cells to specific organs that express the ligand, CXCL12, for this receptor and plays an important role in tumor growth, invasion, metastasis and angiogenesis. In the present study, the inflammatory cytokine, tumor necrosis factor-α (TNF-α) upregulated CXCR4 expression in neuroblastoma cells and increased migration to the CXCR4 ligand SDF-1α. In addition, this effect was dependent upon NF-κB transcriptional activity, as blocking the NF-κB pathway with pyrrolidinedithiocarbamic acid ammonium salt suppressed TNF-α-induced upregulation of CXCR4 expression and reduced the migration towards the CXCR4 ligand, SDF-1α. Treating neuroblastoma cells with TNF-α resulted in the activation of nuclear factor-kappa B (NF-κB) and subsequently, the translocation of NF-κB from the cytoplasm to the nucleus. Using immunohistochemistry, NF-κB and CXCR4 were significantly correlated with each other (P=0.0052, Fisher’s exact test) in a cohort of neuroblastoma samples (n=80). The present study indicates that the inflammatory cytokine, TNF-α, partially functions through the NF-κB signaling pathway to upregulate CXCR4 expression to foster neuroblastoma cell metastasis. These findings indicate that effective inhibition of neuroblastoma metastasis should be directed against the inflammatory cytokine-induced NF-κB/CXCR4/SDF-1α signaling pathway.

24Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors 25 and antibiotic resistance. This transfer is often facilitated via Type 4 Secretion Systems (T4SS), 26 which frequently are encoded on conjugative plasmids. However, donor cells that already contain 27 a particular conjugative plasmid resist acquisition of a 2 nd copy of said plasmid. They utilize 28 different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, 29 encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a 30 role in both virulence and surface exclusion, but the mechanism by which this is achieved has not 31 been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA 32 protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. 33In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby 34 reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the 35 uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA 36 with mutations in the catalytic site we show that the surface exclusion effect is dependent on the 37 activity of the protease domain of PrgA. In silico analysis suggest that PrgA can interact with 38 another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong 39 virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, 40competition mating experiments show that PrgA provides a significant fitness advantage to 41 plasmid-carrying cells. 42 43

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Infections caused by enterohemorrhagic Escherichia coli (EHEC) can lead to diarrhea with abdominal cramps and sometimes are complicated by severe hemolytic uremic syndrome. EHEC secretes effector proteins into host cells through a type III secretion system that is composed of proteins encoded by a chromosomal island, locus for the enterocyte effacement (LEE). EspA is the major component of the filamentous structure connecting the bacteria and the host's cells. Synthesis and secretion of EspA must be carefully controlled since the protein is prone to polymerize. CesAB, CesA2, and EscL have been identified as being able to interact with EspA. Furthermore, the intracellular level of EspA declines when cesAB, cesA2, and escL are individually deleted. Here, we report a LEE gene named l0033, which also affects the intracellular level of EspA. We renamed l0033 as escA since its counterpart in enteropathogenic E. coli has been recently described. Similar to CesAB, EscL, and CesA2, EscA interacts with EspA and enhances the protein stability of EspA. However, EscA is also able to interact with inner membrane-associated EscL, CesA2, and EscN, but not with cytoplasmic CesAB. In terms of gene organizations, escA locates in LEE3. Expression of EscA is faithfully regulated via Mpc, the first gene product of LEE3. Since Mpc is tightly regulated to low level, we suggest that EscA is highly synchronized and critical to the process of escorting EspA to its final destination.