Olga Zaborina

Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1

During host injury, Pseudomonas aeruginosa can be cued to express a lethal phenotype within the intestinal tract reservoir—a hostile, nutrient scarce environment depleted of inorganic phosphate. Here we determined if phosphate depletion activates a lethal phenotype in P. aeruginosa during intestinal colonization. To test this, we allowed Caenorhabditis elegans to feed on lawns of P. aeruginosa PAO1 grown on high and low phosphate media. Phosphate depletion caused PAO1 to kill 60% of nematodes whereas no worms died on high phosphate media. Unexpectedly, intense redness was observed in digestive tubes of worms before death. Using a combination of transcriptome analyses, mutants, and reporter constructs, we identified 3 global virulence systems that were involved in the “red death” response of P. aeruginosa during phosphate depletion; they included phosphate signaling (PhoB), the MvfR–PQS pathway of quorum sensing, and the pyoverdin iron acquisition system. Activation of all 3 systems was required to form a red colored PQS+Fe3+ complex which conferred a lethal phenotype in this model. When pyoverdin production was inhibited in P. aeruginosa by providing excess iron, red death was attenuated in C. elegans and mortality was decreased in mice intestinally inoculated with P. aeruginosa. Introduction of the red colored PQS+Fe3+ complex into the digestive tube of C. elegans or mouse intestine caused mortality associated with epithelial disruption and apoptosis. In summary, red death in C. elegans reveals a triangulated response between PhoB, MvfR–PQS, and pyoverdin in response to phosphate depletion that activates a lethal phenotype in P. aeruginosa.

Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer

The use of live bacteria in the treatment of cancer has a long and interesting history. We report the use of a purified bacterial redox protein, azurin, that enters human cancer (melanoma UISO-Mel-2) cells and induces apoptosis. The induction of apoptosis occurs readily in melanoma cells harboring a functional tumor suppressor protein p53, but much less efficiently in p53-null mutant melanoma (UISO-Mel-6) cells. A redox-negative mutant form of azurin (M44K/M64E) demonstrates much less cytotoxicity to the UISO-Mel-2 cells than the wild-type protein. Azurin has been shown to be internalized in UISO-Mel-2 cells and is localized predominantly in the cytosol and in the nuclear fraction. In the p53-null UISO-Mel-6 cells, azurin is localized only in the cytosol. Thus, intracellular trafficking of azurin to the nucleus is p53-dependent. Azurin forms a complex with p53, thereby stabilizing it and raising its intracellular level in cytosolic, mitochondrial, and nuclear fractions. Corresponding to an increasing level of p53, an inducer of apoptosis, the level of Bax also increases in mitochondria, allowing significant release of mitochondrial cytochrome c into the cytosol, thus initiating the onset of apoptosis. The M44K/M64E mutant form of azurin, deficient in cytotoxicity, is also deficient in forming a complex with p53 and is less efficient in stabilizing p53 than wild-type azurin. Azurin has been shown to allow regression of human UISO-Mel-2 tumors xenotransplanted in nude mice and may potentially be used in cancer treatment.

Membership and Behavior of Ultra-Low-Diversity Pathogen Communities Present in the Gut of Humans during Prolonged Critical Illness

ABSTRACT We analyzed the 16S rRNA amplicon composition in fecal samples of selected patients during their prolonged stay in an intensive care unit (ICU) and observed the emergence of ultra-low-diversity communities (1 to 4 bacterial taxa) in 30% of the patients. Bacteria associated with the genera Enterococcus and Staphylococcus and the family Enterobacteriaceae comprised the majority of these communities. The composition of cultured species from stool samples correlated to the 16S rRNA analysis and additionally revealed the emergence of Candida albicans and Candida glabrata in ~75% of cases. Four of 14 ICU patients harbored 2-member pathogen communities consisting of one Candida taxon and one bacterial taxon. Bacterial members displayed a high degree of resistance to multiple antibiotics. The virulence potential of the 2-member communities was examined in C. elegans during nutrient deprivation and exposure to opioids in order to mimic local conditions in the gut during critical illness. Under conditions of nutrient deprivation, the bacterial members attenuated the virulence of fungal members, leading to a “commensal lifestyle.” However, exposure to opioids led to a breakdown in this commensalism in 2 of the ultra-low-diversity communities. Application of a novel antivirulence agent (phosphate-polyethylene glycol [Pi-PEG]) that creates local phosphate abundance prevented opioid-induced virulence among these pathogen communities, thus rescuing the commensal lifestyle. To conclude, the gut microflora in critically ill patients can consist of ultra-low-diversity communities of multidrug-resistant pathogenic microbes. Local environmental conditions in gut may direct pathogen communities to adapt to either a commensal style or a pathogenic style. IMPORTANCE During critical illness, the normal gut microbiota becomes disrupted in response to host physiologic stress and antibiotic treatment. Here we demonstrate that the community structure of the gut microbiota during prolonged critical illness is dramatically changed such that in many cases only two-member pathogen communities remain. Most of these ultra-low-membership communities display low virulence when grouped together (i.e., a commensal lifestyle); individually, however, they can express highly harmful behaviors (i.e., a pathogenic lifestyle). The commensal lifestyle of the whole community can be shifted to a pathogenic one in response to host factors such as opioids that are released during physiologic stress and critical illness. This shift can be prevented by using compounds such as Pi-PEG15-20 that interrupt bacterial virulence expression. Taking the data together, this report characterizes the plasticity seen with respect to the choice between a commensal lifestyle and a pathogenic lifestyle among ultra-low-diversity pathogen communities that predominate in the gut during critical illness and offers novel strategies for prevention of sepsis. During critical illness, the normal gut microbiota becomes disrupted in response to host physiologic stress and antibiotic treatment. Here we demonstrate that the community structure of the gut microbiota during prolonged critical illness is dramatically changed such that in many cases only two-member pathogen communities remain. Most of these ultra-low-membership communities display low virulence when grouped together (i.e., a commensal lifestyle); individually, however, they can express highly harmful behaviors (i.e., a pathogenic lifestyle). The commensal lifestyle of the whole community can be shifted to a pathogenic one in response to host factors such as opioids that are released during physiologic stress and critical illness. This shift can be prevented by using compounds such as Pi-PEG15-20 that interrupt bacterial virulence expression. Taking the data together, this report characterizes the plasticity seen with respect to the choice between a commensal lifestyle and a pathogenic lifestyle among ultra-low-diversity pathogen communities that predominate in the gut during critical illness and offers novel strategies for prevention of sepsis.

Secretion of ATP-utilizing enzymes, nucleoside diphosphate kinase and ATPase, by Mycobacterium bovis BCG: sequestration of ATP from macrophage P2Z receptors?

Mycobacterium bovis BCG secretes two ATP‐scavenging enzymes, nucleoside diphosphate kinase (Ndk) and ATPase, during growth in Middlebrook 7H9 medium. In synthetic Sauton medium without any protein supplements, there is less secretion of these two enzymes unless proteins such as bovine serum albumin (BSA), ovalbumin or extracts of macrophages are added to the medium. There is a gradient of activity among various proteins in triggering the induction of secretion of these two enzymes. Other mycobacteria, such as M. smegmatis, primarily secrete Ndk, while M. chelonae does not appear to secrete either of these two enzymes. Purification of the enzymes from the culture filtrate of 7H9‐grown M. bovis BCG cells and determination of the N‐terminal amino‐acid sequence have demonstrated a high level of sequence identity of one of the ATPases with DnaK, a heat shock chaperone, of M. tuberculosis and M. leprae, while that of Ndk shows significant identity with the Ndk of Myxococcus xanthus. As both Ndk and ATPase use ATP as a substrate, the physiological significance of the secretion of these two ATP‐utilizing enzymes was explored. External ATP is important in the activation of macrophage surface‐associated P2Z receptors, whose activation has been postulated to allow phagosome–lysosome fusion and macrophage cell death. We demonstrate that the presence of the filtrate containing these enzymes prevents ATP‐induced macrophage cell death, as measured by the release of an intracellular enzyme, lactate dehydrogenase. In vitro complexation studies with purified Ndk/ATPase and hyperproduced P2Z receptor protein will demonstrate whether these enzymes may be used by mycobacteria to sequester ATP from the macrophage P2Z receptors, thereby preventing phagosome–lysosome fusion or macrophage apoptotic death.

The impact of stress and nutrition on bacterial-host interactions at the intestinal epithelial surface.

Purpose of reviewMost literature that examines gut barrier function focuses on alterations in bacterial flora, changes in mucosal epithelium, or the integrity of the mucosal defenses. This review examines new concepts on the interaction between bacteria and the host, the complex relationships that serve to benefit both in times of health, and the alterations and responses that occur during illness. Recent findingsRecent work has demonstrated a more complex relationship between bacteria and barrier integrity and between bacteria themselves, which creates a symbiotic relationship beneficial to both host and flora. Host responses alter this balance, inducing changes in bacteria that may be deleterious to both. SummaryWith a better understanding of the bacteria–host interactions in health and the alterations induced by critical illness, new therapies that improve the environment of both may lead to better recovery rates in intensive care unit patients.

The Bacterial Redox Protein Azurin Induces Apoptosis in J774 Macrophages through Complex Formation and Stabilization of the Tumor Suppressor Protein p53

ABSTRACT Two redox proteins, azurin and cytochrome c551 elaborated by Pseudomonas aeruginosa, demonstrate significant cytotoxic activity towards macrophages. Azurin can enter macrophages, localize in the cytosol and nuclear fractions, and induce apoptosis. Two redox-negative mutants of azurin have less cytotoxicity than does wild-type (wt) azurin. Azurin has been shown to form a complex with the tumor suppressor protein p53, a known inducer of apoptosis, thereby stabilizing it and enhancing its intracellular level. A higher level of reactive oxygen species (ROS), generated during treatment of macrophages with wt azurin, correlates with its cytotoxicity. Treatment with some ROS-removing antioxidants greatly reduces azurin-mediated cytotoxicity, thus demonstrating a novel virulence property of this bacterial redox protein.

Depletion of intestinal phosphate after operative injury activates the virulence of P aeruginosa causing lethal gut-derived sepsis

BACKGROUND We explored the possibility that the opportunistic pathogen, Pseudomonas aeruginosa senses low phosphate (Pi) as a signal of host injury and shifts to a lethal phenotype. METHODS Virulence expression in P aeruginosa was examined in vitro under low phosphate conditions by assessing expression of the PA-I lectin, a barrier dysregulating protein, pyocyanin, and biofilm production, and PstS, a phosphate scavenging protein. Virulence expression in vivo was assessed using operatively injured mice (30% hepatectomy) intestinally inoculated with P aeruginosa. RESULTS In vitro experiments demonstrated that acute phosphate depletion resulted in an increase (P = .001) in the expression the PA-I lectin, biofilm, pyocyanin, and PstS. Operative injury caused a depletion (P = .006) of intestinal phosphate concentration and increased mortality (60%) owing to intestinal P aeruginosa, which was prevented completely with oral phosphate supplementation and restoration of intestinal phosphate, neither of which were observed with systemic (IV) administration. PstS gene expression was 32-fold higher in P aeruginosa recovered from the cecum after hepatectomy indicating inadequate intestinal Pi. CONCLUSIONS Operative injury-induced intestinal phosphate depletion shifts the phenotype of P aeruginosa to express enhanced virulence in vitro and lethality in vivo. Intestinal phosphate repletion may be a novel strategy to contain pathogens associated with lethal gut-derived sepsis.

Collagen degradation and MMP9 activation by Enterococcus faecalis contribute to intestinal anastomotic leak

Enterococcus faecalis depletes intestinal collagen, activates the host tissue protease MMP9, and contributes to anastomotic leak. Can our gut microbes prevent wound healing? In a new study, Shogan et al. examined whether the bacterium Enterococcus faecalis, normally present in the intestine, contributes to anastomotic leak, the most feared complication after intestinal surgery. They demonstrated that intestinal E. faecalis can produce a tissue-destroying enzyme that affects the normal healing process by breaking down collagen, a protein that is critical to fully seal the intestine after its removal and reconnection. E. faecalis also activates a host gut enzyme, MMP9, further contributing to anastomotic leak. Finally, the authors demonstrated that the most common antibiotic used in intestinal surgery does not eliminate E. faecalis and thus does not prevent anastomotic leak. 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.

Pseudomonas aeruginosa expresses a lethal virulence determinant, the PA-I lectin/adhesin, in the intestinal tract of a stressed host: The role of epithelia cell contact and molecules of the quorum sensing signaling system

Objective: We have previously demonstrated that P. aeruginosa can have profound effects on the intestinal epithelial barrier via one of its virulence factors, the PA-I lectin/adhesin. The aims of the present study were to further characterize the interaction of P. aeruginosa and the intestinal epithelium using both in vitro and in vivo approaches Methods: In vitro assays examining the effect of bacterial growth phase, epithelial cell contact, and butanoyl homoserine lactone (C4-HSL), a quorum sensing signaling molecule know to affect various extracellular virulence factors in P. aeruginosa, on PA-I expression in P. aeruginosa were performed. In vivo studies were carried out by modeling catabolic stress in mice using a 30% surgical hepatectomy and direct introduction of P. aeruginosa and various virulence components into the cecum. The effect of this model on PA-I expression in P. aeruginosa was determined Results: Results demonstrated that PA-I expression in P. aeruginosa is affected by its phase of growth, its contact to the intestinal epithelium, and its exposure to the quorum sensing molecule, C4-HSL. Furthermore, data from the present study suggest that the PA-I lectin/adhesin of P. aeruginosa may be increased in vivo by local factors within the cecum of mice in response to surgical stress Conclusions: These data indicate that multiple factors present in the intestinal microenvironment of a stressed host may induce certain opportunistic pathogens to express key virulence factors leading to a state of lethal gut-derived sepsis.

Trauma/SepsisDepletion of intestinal phosphate after operative injury activates the virulence of P aeruginosa causing lethal gut-derived sepsis

BACKGROUND We explored the possibility that the opportunistic pathogen, Pseudomonas aeruginosa senses low phosphate (Pi) as a signal of host injury and shifts to a lethal phenotype. METHODS Virulence expression in P aeruginosa was examined in vitro under low phosphate conditions by assessing expression of the PA-I lectin, a barrier dysregulating protein, pyocyanin, and biofilm production, and PstS, a phosphate scavenging protein. Virulence expression in vivo was assessed using operatively injured mice (30% hepatectomy) intestinally inoculated with P aeruginosa. RESULTS In vitro experiments demonstrated that acute phosphate depletion resulted in an increase (P = .001) in the expression the PA-I lectin, biofilm, pyocyanin, and PstS. Operative injury caused a depletion (P = .006) of intestinal phosphate concentration and increased mortality (60%) owing to intestinal P aeruginosa, which was prevented completely with oral phosphate supplementation and restoration of intestinal phosphate, neither of which were observed with systemic (IV) administration. PstS gene expression was 32-fold higher in P aeruginosa recovered from the cecum after hepatectomy indicating inadequate intestinal Pi. CONCLUSIONS Operative injury-induced intestinal phosphate depletion shifts the phenotype of P aeruginosa to express enhanced virulence in vitro and lethality in vivo. Intestinal phosphate repletion may be a novel strategy to contain pathogens associated with lethal gut-derived sepsis.