Andrew Y. Koh

Mucosal damage and neutropenia are required for Candida albicans dissemination

Candida albicans fungemia in cancer patients is thought to develop from initial gastrointestinal (GI) colonization with subsequent translocation into the bloodstream after administration of chemotherapy. It is unclear what components of the innate immune system are necessary for preventing C. albicans dissemination from the GI tract, but we have hypothesized that both neutropenia and GI mucosal damage are critical for allowing widespread invasive C. albicans disease. We investigated these parameters in a mouse model of C. albicans GI colonization that led to systemic spread after administration of immunosuppression and mucosal damage. After depleting resident GI intestinal flora with antibiotic treatment and achieving stable GI colonization levels of C. albicans, it was determined that systemic chemotherapy with cyclophosphamide led to 100% mortality, whereas selective neutrophil depletion, macrophage depletion, lymphopenia or GI mucosal disruption alone resulted in no mortality. Selective neutrophil depletion combined with GI mucosal disruption led to disseminated fungal infection and 100% mortality ensued. GI translocation and dissemination by C. albicans was also dependent on the organisms ability to transform from the yeast to the hyphal form. This mouse model of GI colonization and fungemia is useful for studying factors of innate host immunity needed to prevent invasive C. albicans disease as well as identifying virulence factors that are necessary for fungal GI colonization and dissemination. The model may also prove valuable for evaluating therapies to control C. albicans infections.

Inescapable Need for Neutrophils as Mediators of Cellular Innate Immunity to Acute Pseudomonas aeruginosa Pneumonia

ABSTRACT Pseudomonas aeruginosa is a leading cause of pneumonia, and many components of the innate immune system have been proposed to exert important effects in preventing lung infection. However, a vigorous experimental system to identify an overriding, key effector mediating innate immunity to lung infection has not been utilized. As many of the important components of innate immunity are involved in recruitment and activation of polymorphonuclear neutrophils (PMNs) to infected tissues, we hypothesized that the cells and factors needed for their proper recruitment to the lung comprised the major mediators of innate immunity. In neutropenic mice, intranasal (i.n.) doses of P. aeruginosa as low as 10 to 100 CFU/mouse produced a fatal lung infection, compared with 107 to >108 CFU for nonneutropenic mice. There was only a very modest increased mortality in mice lacking mature lymphocytes and no increased mortality in mice depleted of alveolar macrophages when administered i.n. P. aeruginosa. Recombinant mouse granulocyte colony-stimulating factor increased survival of neutropenic mice after i.n. P. aeruginosa inoculation. MyD88−/− mice, which cannot recruit PMNs to the lungs, were highly susceptible to fatal P. aeruginosa lung infection, with bacterial doses of <120 CFU being lethal. Activation of a MyD88-independent pathway for PMN recruitment to the lungs in MyD88−/− mice resulted in enhanced protection against P. aeruginosa lung infection. Overall, in the absence of PMNs, mice cannot resist P. aeruginosa lung infection from extremely small bacterial doses. There is an inescapable requirement for local PMN recruitment and activation to mediate innate immunity to P. aeruginosa lung infection.

Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization

Candida albicans colonization is required for invasive disease. Unlike humans, adult mice with mature intact gut microbiota are resistant to C. albicans gastrointestinal (GI) colonization, but the factors that promote C. albicans colonization resistance are unknown. Here we demonstrate that commensal anaerobic bacteria—specifically clostridial Firmicutes (clusters IV and XIVa) and Bacteroidetes—are critical for maintaining C. albicans colonization resistance in mice. Using Bacteroides thetaiotamicron as a model organism, we find that hypoxia-inducible factor-1α (HIF-1α), a transcription factor important for activating innate immune effectors, and the antimicrobial peptide LL-37 (CRAMP in mice) are key determinants of C. albicans colonization resistance. Although antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease. In the setting of antibiotics, Hif1a and Camp (which encodes CRAMP) are required for B. thetaiotamicron–induced protection against C. albicans colonization of the gut. Thus, modulating C. albicans GI colonization by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

Virulence of Pseudomonas aeruginosa in a murine model of gastrointestinal colonization and dissemination in neutropenia.

ABSTRACT Pseudomonas aeruginosa bacteremia in cancer patients develops from initial gastrointestinal (GI) colonization with translocation into the bloodstream in the setting of chemotherapy-induced neutropenia and GI mucosal damage. We established a reproducible mouse model of P. aeruginosa GI colonization and systemic spread during neutropenia. Mice received 2 mg of streptomycin/ml of drinking water and 1,500 U of penicillin G/ml for 4 days and then ingested 107 CFU of P. aeruginosa per ml of drinking water for 5 days. After GI colonization levels were determined, cyclophosphamide (Cy) was then injected intraperitoneally (i.p.) three times every other day or an antineutrophil monoclonal antibody, RB6-8C5, was injected i.p. once. Dissemination was defined by the presence of P. aeruginosa in spleens of moribund or dead mice. In this mouse model, P. aeruginosa colonizes the GI tract and then disseminates systemically once Cy or RB6-8C5 is administered. The duration and intensity of neutropenia, related to Cy dose, was found to be a means to compare the virulence of different P. aeruginosa strains, as exhibited by comparisons of strains lacking or producing the virulence-enhancing ExoU cytotoxin. The lipopolysaccharide outer core polysaccharide and O side chains were critical in establishing GI colonization, and P. aeruginosa mutants lacking the aroA gene (necessary for synthesizing aromatic amino acids) were able to establish GI colonization but unable to disseminate. Both the colonization and dissemination phases of P. aeruginosa pathogenesis can be studied in this model, which should prove useful for evaluating pathogenesis, therapies, and associated means to control P. aeruginosa nosocomial infections.

Murine models of Candida gastrointestinal colonization and dissemination.

ABSTRACT Ninety-five percent of infectious agents enter through exposed mucosal surfaces, such as the respiratory and gastrointestinal (GI) tracts. The human GI tract is colonized with trillions of commensal microbes, including numerous Candida spp. Some commensal microbes in the GI tract can cause serious human infections under specific circumstances, typically involving changes in the gut environment and/or host immune conditions. Therefore, utilizing animal models of fungal GI colonization and dissemination can lead to significant insights into the complex pathophysiology of transformation from a commensal organism to a pathogen and host-pathogen interactions. This paper will review the methodologic approaches used for modeling GI colonization versus dissemination, the insights learned from these models, and finally, possible future directions using these animal modeling systems.

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa’s ability to colonize the GI tract but does decrease P. aeruginosa’s cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.

Precision editing of the gut microbiota ameliorates colitis

Inflammatory diseases of the gastrointestinal tract are frequently associated with dysbiosis, characterized by changes in gut microbial communities that include an expansion of facultative anaerobic bacteria of the Enterobacteriaceae family (phylum Proteobacteria). Here we show that a dysbiotic expansion of Enterobacteriaceae during gut inflammation could be prevented by tungstate treatment, which selectively inhibited molybdenum-cofactor-dependent microbial respiratory pathways that are operational only during episodes of inflammation. By contrast, we found that tungstate treatment caused minimal changes in the microbiota composition under homeostatic conditions. Notably, tungstate-mediated microbiota editing reduced the severity of intestinal inflammation in mouse models of colitis. We conclude that precision editing of the microbiota composition by tungstate treatment ameliorates the adverse effects of dysbiosis in the inflamed gut.

Metagenomic Shotgun Sequencing and Unbiased Metabolomic Profiling Identify Specific Human Gut Microbiota and Metabolites Associated with Immune Checkpoint Therapy Efficacy in Melanoma Patients

This is the first prospective study of the effects of human gut microbiota and metabolites on immune checkpoint inhibitor (ICT) response in metastatic melanoma patients. Whereas many melanoma patients exhibit profound response to ICT, there are fewer options for patients failing ICT—particularly with BRAF-wild-type disease. In preclinical studies, specific gut microbiota promotes regression of melanoma in mice. We therefore conducted a study of the effects of pretreatment gut microbiota and metabolites on ICT Response Evaluation Criteria in Solid Tumors response in 39 metastatic melanoma patients treated with ipilimumab, nivolumab, ipilimumab plus nivolumab (IN), or pembrolizumab (P). IN yielded 67% responses and 8% stable disease; P achieved 23% responses and 23% stable disease. ICT responders for all types of therapies were enriched for Bacteroides caccae. Among IN responders, the gut microbiome was enriched for Faecalibacterium prausnitzii, Bacteroides thetaiotamicron, and Holdemania filiformis. Among P responders, the microbiome was enriched for Dorea formicogenerans. Unbiased shotgun metabolomics revealed high levels of anacardic acid in ICT responders. Based on these pilot studies, both additional confirmatory clinical studies and preclinical testing of these bacterial species and metabolites are warranted to confirm their ICT enhancing activity.

Intravenous Pentamidine Is Safe and Effective as Primary Pneumocystis Pneumonia Prophylaxis in Children and Adolescents Undergoing Hematopoietic Stem Cell Transplantation

Background: Pneumocystis carinii pneumonia (PCP) is a potentially life-threatening but preventable infection that may occur after hematopoietic stem cell transplantation (HSCT). Intravenous pentamidine has been used in the prevention of PCP in the post-transplant period, although there are few trials published in the literature evaluating its safety and efficacy. Methods: We retrospectively reviewed the medical records of children who underwent HSCT from January 1, 2005, to October 1, 2011, who received intravenous pentamidine as first-line PCP prophylaxis initiated at admission. Demographic, clinical, microbiologic, management and outcome data were collected. Results: One hundred sixty-seven consecutive HSCTs in 137 pediatric patients were given intravenous pentamidine before myeloablation and then every 28 days until the subject was at least a minimum 30 days post-HSCT, had stable neutrophil engraftment (absolute neutrophil count >1000/mm2 for 3 days without growth factor support) and for allogeneic patients, no evidence of active graft versus host disease and weaning on their immunosuppressive therapy. No cases of PCP were seen in this cohort. Ten (7%) had a grade I side effect of nausea/vomiting requiring slower infusion time and 2 (2%) had a grade IV reaction with anaphylaxis (rash) and hypotension with 1 child requiring transfer to the intensive care unit. Conclusions: Intravenous pentamidine was safe and effective for the prevention of PCP in pediatric HSCT patients. Given the potential neutropenic effects of trimethoprim-sulfamethoxazole, compliance with drug administration and inferior efficacy of other PCP prophylactic medications, intravenous pentamidine should be considered as first-line therapy for the prevention of PCP in children undergoing HSCT.

Utility of in vivo transcription profiling for identifying Pseudomonas aeruginosa genes needed for gastrointestinal colonization and dissemination.

Microarray analysis of Pseudomonas aeruginosa mRNA transcripts expressed in vivo during animal infection has not been previously used to investigate potential virulence factors needed in this setting. We compared mRNA expression in bacterial cells recovered from the gastrointestinal (GI) tracts of P. aeruginosa-colonized mice to that of P. aeruginosa in the drinking water used to colonize the mice. Genes associated with biofilm formation and type III secretion (T3SS) had markedly increased expression in the GI tract. A non-redundant transposon library in P. aeruginosa strain PA14 was used to test mutants in genes identified as having increased transcription during in vivo colonization. All of the Tn-library mutants in biofilm-associated genes had an attenuated ability to form biofilms in vitro, but there were no significant differences in GI colonization and dissemination between these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI colonization among these mutant strains and their WT counterparts, whereas rates of survival following dissemination were significantly decreased in mice infected by the T3SS mutant strains. However, there was a variable, strain-dependent effect on overall survival between parental and T3SS mutants. Thus, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia.