A commensal-encoded genotoxin drives restriction of Vibrio cholerae colonization and host gut microbiome remodeling.

SignificanceIn a polymicrobial battlefield where different species compete for nutrients and colonization niches, antimicrobial compounds are the sword and shield of commensal microbes in competition with invading pathogens and each other. The identification of an Escherichia coli-produced genotoxin, colibactin, and its specific targeted killing of enteric pathogens and commensals, including Vibrio cholerae and Bacteroides fragilis, sheds light on our understanding of intermicrobial interactions in the mammalian gut. Our findings elucidate the mechanisms through which genotoxins shape microbial communities and provide a platform for probing the larger role of enteric multibacterial interactions regarding infection and disease outcomes.

S-Nitrosylation of the virulence regulator AphB promotes Vibrio cholerae pathogenesis.

Vibrio cholerae is the etiologic agent of the severe human diarrheal disease cholera. To colonize mammalian hosts, this pathogen must defend against host-derived toxic compounds, such as nitric oxide (NO) and NO-derived reactive nitrogen species (RNS). RNS can covalently add an NO group to a reactive cysteine thiol on target proteins, a process called protein S-nitrosylation, which may affect bacterial stress responses. To better understand how V. cholerae regulates nitrosative stress responses, we profiled V. cholerae protein S-nitrosylation during RNS exposure. We identified an S-nitrosylation of cysteine 235 of AphB, a LysR-family transcription regulator that activates the expression of tcpP, which activates downstream virulence genes. Previous studies show that AphB C235 is sensitive to O2 and reactive oxygen species (ROS). Under microaerobic conditions, AphB formed dimer and directly repressed transcription of hmpA, encoding a flavohemoglobin that is important for NO resistance of V. cholerae. We found that tight regulation of hmpA by AphB under low nitrosative stress was important for V. cholerae optimal growth. In the presence of NO, S-nitrosylation of AphB abolished AphB activity, therefore relieved hmpA expression. Indeed, non-modifiable aphBC235S mutants were sensitive to RNS in vitro and drastically reduced colonization of the RNS-rich mouse small intestine. Finally, AphB S-nitrosylation also decreased virulence gene expression via debilitation of tcpP activation, and this regulation was also important for V. cholerae RNS resistance in vitro and in the gut. These results suggest that the modulation of the activity of virulence gene activator AphB via NO-dependent protein S-nitrosylation is critical for V. cholerae RNS resistance and colonization.

Siderophore piracy enhances Vibrio cholerae environmental survival and pathogenesis.

Vibrio cholerae, the aetiological agent of cholera, possesses multiple iron acquisition systems, including those for the transport of siderophores. How these systems benefit V. cholerae in low-iron, polymicrobial communities in environmental settings or during infection remains poorly understood. Here, we demonstrate that in iron-limiting conditions, co-culture of V. cholerae with a number of individual siderophore-producing microbes significantly promoted V. cholerae growth in vitro. We further show that in the host environment with low iron, V. cholerae colonizes better in adult mice in the presence of the siderophore-producing commensal Escherichia coli. Taken together, our results suggest that in aquatic reservoirs or during infection, V. cholerae may overcome environmental and host iron restriction by hijacking siderophores from other microbes.

Pretransplant Hepatic Malignancy Increases Risk of De Novo Malignancy after Liver Transplantation.

BACKGROUND: Hepatocellular carcinoma (HCC) recurrence and development of de novo malignancy (DNM) after liver transplantation (LT) are the major causes of late recipient death. METHODS: We analyzed the incidence of extrahepatic DNM following living donor LT according to the status of pretransplant hepatic malignancy. We selected 2,076 adult patients who underwent primary LDLT during 7 years from January 2010 to December 2016. RESULTS: The pretransplant hepatic malignancy group (n = 1,012) showed 45 cases (4.4%) of the following extrahepatic DNMs: posttransplant lymphoproliferative disease (PTLD) in 10; lung cancer in 10; stomach cancer in 6; colorectal cancer in 5; urinary bladder cancer in 3; and other cancers in 11. The pretransplant no hepatic malignancy group (n = 1,064) showed 25 cases (2.3%) of the following extrahepatic DNMs: colorectal cancer in 3; stomach cancer in 3; leukemia in 3; lung cancer in 3; PTLD in 2; prostate cancer in 2; and other cancers in 9. Incidences of extrahepatic DNM in the pretransplant hepatic malignancy and no hepatic malignancy groups were as follows: 1.1% and 0.5% at 1 year, 3.2% and 2.0% at 3 years, 4.6% and 2.5% at 5 years, and 5.4% and 2.8% at 8 years, respectively (P = 0.006). Their overall patient survival rates were as follows: 97.3% and 97.2% at 1 year, 91.6% and 95.9% at 3 years, 89.8% and 95.4% at 5 years, and 89.2% and 95.4% at 8 years, respectively (P < 0.001). Pretransplant hepatic malignancy was the only significant risk factor for posttransplant extrahepatic DNM. CONCLUSION: Our results suggest that patients who had pretransplant hepatic malignancy be followed up more strictly because they have a potential risk of primary hepatic malignancy recurrence as well as a higher risk of extrahepatic DNM than patients without pretransplant hepatic malignancy.

Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO(3+), for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1.

The enzyme cytochrome P450 11A1 cleaves the C20-C22 carbon-carbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carbon-carbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound I (FeO(3+)). Compound I can either (i) abstract an O-H hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol. The mechanism of this carbon-carbon bond cleavage step was tested using (18)O-labeled molecular oxygen and purified P450 11A1. P450 11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of molecular oxygen ((18)O2), and coupled assays were used to trap the labile (18)O atoms in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting products were derivatized and the (18)O content was analyzed by high-resolution mass spectrometry. P450 11A1 showed no incorporation of an (18)O atom into either of its carbon-carbon bond cleavage products, pregnenolone and isocaproaldehyde . The positive control experiments established retention of the carbonyl oxygens in the enzymatic products during the trapping and derivatization processes. These results reveal a mechanism involving an electrophilic Compound I species that reacts with nucleophilic hydroxy groups in the 20R,22R-dihydroxycholesterol intermediate of the P450 11A1 reaction to produce the key steroid pregnenolone.

Long-term patency and complications of ringed polytetrafluoroethylene grafts used for middle hepatic vein reconstruction in living-donor liver transplantation.

Background: Homologous vein allografts are adequate for reconstruction of the middle hepatic vein (MHV) in living-donor liver transplantation (LDLT). However, supply is a matter of concern. To replace homologous vein allografts, polytetrafluoroethylene (PTFE) grafts were used. This study aimed to assess the long-term patency rates and complications of PTFE grafts used for MHV reconstruction of LDLT in a high-volume liver transplantation center. Methods: We analyzed the patency rates of PTFE-interposed MHV in 100 LDLT recipients and reviewed complications including PTFE graft migration. Results: The mean age was 53.5±5.4 years and male to female ratio was 73:27. Primary diagnoses were hepatitis B virus infection (n=71) and other (n=28). Mean model for end-stage liver disease score was 16.2±8.3. V5 reconstruction was performed as either single anastomosis (n=85) or double anastomoses (n=14). No V5 reconstruction was required in one patient. V8 reconstruction was performed as single anastomosis, double anastomoses, and no reconstruction in 75, 0, and 25 patients, respectively. During a mean follow-up of 6 years, three recipients required early MHV stenting within 2 weeks. After 3 months, there were no episodes of congestion-associated infarct, regardless of MHV patency. Patency rates of PTFE-interposed MHV were 54.0%, 37.0%, and 37.0% at 1, 3, and 5 years, respectively. Unwanted PTFE graft migration occurred in two recipients, and the actual incidence was 2% at 5 years. Conclusions: PTFE grafts combined with small-artery patches demonstrated acceptably high short- and long-term patency rates. Since the risk of unwanted migration of PTFE graft is not negligibly low, lifelong surveillance is necessary to detect unexpected rare complications.

Is renal replacement therapy necessary in deceased donor liver transplantation candidates with hepatorenal syndrome?: a 2-year experience at a high-volume center.

PURPOSE: Hepatorenal syndrome (HRS) is a fatal complication in patients with end-stage liver disease awaiting liver transplantation (LT). HRS often develops in patients with high model for end-stage liver disease (MELD) score. This study investigated the outcomes of peritransplant management of HRS in a high-volume LT center in Korea for 2 years. METHODS: A total of 157 recipients that deceased donor liver transplantation (DDLT) from January 2017 to December 2018 were included. In-hospital mortality (IHM) was analyzed in relation to pre- and posttransplant application of renal replacement therapy (RRT). RESULTS: Primary diagnoses for DDLT were alcoholic liver disease (n = 61), HBV-associated liver cirrhosis (n = 48), retransplantation for chronic graft failure (n = 24), and others (n = 24). Mean MELD score was 34.6 ± 6.2 with 72 patients at Korean Network for Organ Sharing MELD status 2 (45.9%), 43 at status 3 (27.4%), 36 at status 4 (22.9%), and 6 at status 5 (3.8%). Pretransplant RRT was performed in 16 patients (10.2%) that did not show IHM. Posttransplant RRT was performed in 69 patients (44.0%), for whom IHM incidence was 15.9%. In 53 patients that had undergone de novo posttransplant RRT, IHM incidence increased to 20.8%. IHM in the 88 patients not requiring RRT was 2.3%. CONCLUSION: The majority of adult DDLT recipients in Korean MELD score-based allocation system have very high MELD scores, which is often associated with HRS. Pretransplant RRT appears to improve posttransplant survival outcomes. We thereby recommend that, if indicated, pretransplant RRT be performed while awaiting DDLT.

Hemashield Vascular Graft Is a Preferable Prosthetic Graft for Middle Hepatic Vein Reconstruction in Living Donor Liver Transplantation.

BACKGROUND Because of the supply shortage for homologous vein allografts, we previously used ringed Gore-Tex vascular grafts for middle hepatic vein (MHV) reconstruction in living donor liver transplantation. However, owing to the subsequent unavailability of ringed Gore-Tex grafts, we replaced them with Hemashield vascular grafts. This study aimed to compare the patency of Hemashield grafts with that of ringed Gore-Tex grafts. MATERIAL AND METHODS This was a retrospective double-arm study between the study group that used Hemashield grafts (n=63) and the historical control group that used ringed Gore-Tex grafts (n=126). RESULTS In the Gore-Tex and Hemashield groups, mean age was 53.1±6.2 and 54.3±10.4 years; model for end-stage liver disease score was 16.5±8.3 and 17.5±9.9; and graft-recipient weight ratio was 1.11±0.23 and 1.12±0.25, respectively. In the Gore-Tex graft group, V5 reconstruction was done in single (n=107, 84.9%), double (n=17, 13.5%), and none (n=2, 1.6%). V8 reconstruction was done in single (n=95, 75.4%), double (n=1, 0.8%), and none (n=30, 23.8%). In the Hemashield group, V5 reconstruction was done in single (n=43, 68.3%), double (n=19, 30.2%), and triple (n=1, 1.6%). V8 reconstruction was done in single (n=45, 71.4%), double (n=9, 14.3%), and none (n=9, 14.3%). One-year conduit patency rates in the Gore-Tex and Hemashield groups were 54.8% and 71.6%, respectively (p=0.048). CONCLUSIONS MHV reconstruction using Hemashield vascular grafts demonstrated higher short-term patency rates than those associated with ringed Gore-Tex vascular grafts. We suggest that the Hemashield vascular graft is one of the best prosthetic materials for MHV reconstruction.

Gluconeogenic growth of Vibrio cholerae is important for competing with host gut microbiota.

PURPOSE: The gastrointestinal tract is home to thousands of commensal bacterial species. Therefore, competition for nutrients is paramount for successful bacterial pathogen invasion of intestinal ecosystems. The human pathogen Vibrio cholerae, the causative agent of the severe diarrhoeal disease, cholera, is able to colonize the small intestine, which is protected by mucus. However, it is unclear which metabolic pathways or nutrients V. cholerae utilizes during intestinal colonization and growth. METHODOLOGY: In this study, we investigated the effect of various metabolic key genes, including those involved in the gluconeogenesis pathway, on V. cholerae physiology and in vivo colonization. RESULTS: We found that gluconeogenesis is important for infant mouse colonization. Growth assays showed that mutations in the key components of gluconeogenesis pathway, PpsA and PckA, lead to a growth defect in a minimal medium supplemented with mucin as a carbon source. Furthermore, the ppsA/pckA mutants colonized poorly in the adult mouse intestine, particularly when more gut commensal flora are present. CONCLUSION: Gluconeogenesis biosynthesis is important for the successful colonization of V. cholerae in a niche that is full of competing microbiota.