Natalia Belogortseva

Even under the most expert care, a properly constructed intestinal anastomosis can fail to heal, resulting in leakage of its contents, peritonitis, and sepsis. The cause of anastomotic leak remains unknown, and its incidence has not changed in decades. We demonstrate that the commensal bacterium Enterococcus faecalis contributes to the pathogenesis of anastomotic leak through its capacity to degrade collagen and to activate tissue matrix metalloproteinase 9 (MMP9) in host intestinal tissues. We demonstrate in rats that leaking anastomotic tissues were colonized by E. faecalis strains that showed an increased collagen-degrading activity and also an increased ability to activate host MMP9, both of which contributed to anastomotic leakage. We demonstrate that the E. faecalis genes gelE and sprE were required for E. faecalis-mediated MMP9 activation. Either elimination of E. faecalis strains through direct topical antibiotics applied to rat intestinal tissues or pharmacological suppression of intestinal MMP9 activation prevented anastomotic leak in rats. In contrast, the standard recommended intravenous antibiotics used in patients undergoing colorectal surgery did not eliminate E. faecalis at anastomotic tissues nor did they prevent leak in our rat model. Finally, we show in humans undergoing colon surgery and treated with the standard recommended intravenous antibiotics that their anastomotic tissues still contained E. faecalis and other bacterial strains with collagen-degrading/MMP9-activating activity. We suggest that intestinal microbes with the capacity to produce collagenases and to activate host metalloproteinase MMP9 may break down collagen in the intestinal tissue contributing to anastomotic leak.

The key regulators of intracellular trafficking, Ypt/Rab GTPases, are stimulated by specific upstream activators and, when activated, recruit specific downstream effectors to mediate membrane-transport events. The yeast Ypt1 and its human functional homolog hRab1 regulate both endoplasmic reticulum (ER)-to-Golgi transport and autophagy. However, it is not clear whether the mechanism by which these GTPases regulate autophagy depends on their well-documented function in ER-to-Golgi transport. Here, we identify Atg11, the preautophagosomal structure (PAS) organizer, as a downstream effector of Ypt1 and show that the Ypt1–Atg11 interaction is required for PAS assembly under normal growth conditions. Moreover, we show that Ypt1 and Atg11 colocalize with Trs85, a Ypt1 activator subunit, and together they regulate selective autophagy. Finally, we show that Ypt1 and Trs85 interact on Atg9-containing membranes, which serve as a source for the membrane component of the PAS. Together our results define a Ypt/Rab module—comprising an activator, GTPase, and effector—that orchestrates the onset of selective autophagy, a process vital for cell homeostasis. Furthermore, because Atg11 does not play a role in ER-to-Golgi transport, we demonstrate here that Ypt/Rabs can regulate two independent membrane-transport processes by recruiting process-specific effectors.

Chmp1A (Chromatin modifying protein 1A/Charged multivesicular protein 1A) is a member of the ESCRT-III (Endosomal Sorting Complex Required for Transport) family that was shown to function in endosome-mediated trafficking via multivesicular body (MVB) formation and sorting. Recent reports suggest that ESCRT complexes are also involved in cell cycle progression and tumor development. Using in vitro and in vivo model systems, we provide evidence that Chmp1A is a novel tumor suppressor, especially in the pancreas. We demonstrated that short hairpin RNA (shRNA) mediated stable silencing of Chmp1A in HEK 293T cells resulted in an increase of anchorage-independent growth in soft agar assay and tumor formation in xenograft assay. To investigate the involvement of Chmp1A in human tumor development we screened human cancer arrays and pancreatic tissue arrays. We discovered that Chmp1A mRNA and protein was reduced and/or altered (protein) in various human pancreatic tumors. To investigate the biological implication of these data, we either overexpressed or silenced Chmp1A in human pancreatic ductal tumor cells (PanC-1) and studied the effect of these manipulations on cell and tumor growth respectively. Stable overexpression of Chmp1A in PanC-1 cells resulted in cell growth inhibition and tumor xenograft inhibition respectively. In contrast, silencing of Chmp1A in PanC-1 cells resulted in the elevation of cell growth in vitro. Mechanistically, overexpression of Chmp1A strongly increased the protein level of p53 and phospho-P53. Taken together, our data indicates that Chmp1A is a novel tumor suppressor, especially in pancreas and that Chmp1A regulates tumor growth potentially through p53 signaling pathway.

Antibiotic resistance among highly pathogenic strains of bacteria and fungi is a growing concern in the face of the ability to sustain life during critical illness with advancing medical interventions. The longer patients remain critically ill, the more likely they are to become colonized by multidrug-resistant (MDR) pathogens. The human gastrointestinal tract is the primary site of colonization of many MDR pathogens and is a major source of life-threatening infections due to these microorganisms. Eradication measures to sterilize the gut are difficult if not impossible and carry the risk of further antibiotic resistance. Here, we present a strategy to contain rather than eliminate MDR pathogens by using an agent that interferes with the ability of colonizing pathogens to express virulence in response to host-derived and local environmental factors. The antivirulence agent is a phosphorylated triblock high-molecular-weight polymer (here termed Pi-PEG 15-20) that exploits the known properties of phosphate (Pi) and polyethylene glycol 15-20 (PEG 15-20) to suppress microbial virulence and protect the integrity of the intestinal epithelium. The compound is nonmicrobiocidal and appears to be highly effective when tested both in vitro and in vivo. Structure functional analyses suggest that the hydrophobic bis-aromatic moiety at the polymer center is of particular importance to the biological function of Pi-PEG 15-20, beyond its phosphate content. Animal studies demonstrate that Pi-PEG prevents mortality in mice inoculated with multiple highly virulent pathogenic organisms from hospitalized patients in association with preservation of the core microbiome.