Laura Rossi

Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole

Resistance to widely used fungistatic drugs, particularly to the ergosterol biosynthesis inhibitor fluconazole, threatens millions of immunocompromised patients susceptible to invasive fungal infections. The dense network structure of synthetic lethal genetic interactions in yeast suggests that combinatorial network inhibition may afford increased drug efficacy and specificity. We carried out systematic screens with a bioactive library enriched for off‐patent drugs to identify compounds that potentiate fluconazole action in pathogenic Candida and Cryptococcus strains and the model yeast Saccharomyces. Many compounds exhibited species‐ or genus‐specific synergism, and often improved fluconazole from fungistatic to fungicidal activity. Mode of action studies revealed two classes of synergistic compound, which either perturbed membrane permeability or inhibited sphingolipid biosynthesis. Synergistic drug interactions were rationalized by global genetic interaction networks and, notably, higher order drug combinations further potentiated the activity of fluconazole. Synergistic combinations were active against fluconazole‐resistant clinical isolates and an in vivo model of Cryptococcus infection. The systematic repurposing of approved drugs against a spectrum of pathogens thus identifies network vulnerabilities that may be exploited to increase the activity and repertoire of antifungal agents.

The Loss of Topography in the Microbial Communities of the Upper Respiratory Tract in the Elderly

RATIONALE The microbial communities inhabiting the upper respiratory tract protect from respiratory infection. The maturity of the immune system is a major influence on the composition of the microbiome and, in youth, the microbiota and immune system are believed to mature in tandem. With age, immune function declines and susceptibility to respiratory infection increases. Whether these changes contribute to the microbial composition of the respiratory tract is unknown. OBJECTIVES Our goal was to determine whether the microbes of the upper respiratory tract differ between mid-aged adults (18-40 yr) and the elderly (>65 yr). METHODS Microbiomes of the anterior nares and oropharynx of elderly individuals were evaluated by 16S rRNA gene sequencing. These communities were compared with data on mid-aged adults obtained from the Human Microbiome Project. MEASUREMENTS AND MAIN RESULTS The microbiota of the elderly showed no associations with sex, comorbidities, residence, or vaccinations. Comparisons of mid-aged adults and the elderly demonstrated significant differences in the composition of the anterior nares and oropharynx, including a population in the anterior nares of the elderly that more closely resembled the oropharynx than the anterior nares of adults. The elderly oropharyngeal microbiota were characterized by increased abundance of streptococci, specifically, Streptococcus salivarius group species, but not Streptococcus pneumoniae, carriage of which was low (<3% of participants), as demonstrated by PCR (n = 4/123). CONCLUSIONS Microbial populations of the upper respiratory tract in mid-aged adults and the elderly differ; it is possible that these differences contribute to the increased risk of respiratory infections experienced by the elderly.

Role of Homoserine Transacetylase as a New Target for Antifungal Agents

ABSTRACT Microbial amino acid biosynthesis is a proven yet underexploited target of antibiotics. The biosynthesis of methionine in particular has been shown to be susceptible to small-molecule inhibition in fungi. The first committed step in Met biosynthesis is the acylation of homoserine (Hse) by the enzyme homoserine transacetylase (HTA). We have identified the MET2 gene of Cryptococcus neoformans H99 that encodes HTA (CnHTA) by complementation of an Escherichia coli metA mutant that lacks the gene encoding homoserine transsuccinylase (HTS). We cloned, expressed, and purified CnHTA and determined its steady-state kinetic parameters for the acetylation of L-Hse by acetyl coenzyme A. We next constructed a MET2 mutant in C. neoformans H99 and tested its growth behavior in Met-deficient media, confirming the expected Met auxotrophy. Furthermore, we used this mutant in a mouse inhalation model of infection and determined that MET2 is required for virulence. This makes fungal HTA a viable target for new antibiotic discovery. We screened a 1,000-compound library of small molecules for HTA inhibitors and report the identification of the first inhibitor of fungal HTA. This work validates HTA as an attractive drug-susceptible target for new antifungal agent design.

NSAID enteropathy and bacteria: a complicated relationship

The clinical significance of small intestinal damage caused by nonsteroidal anti-inflammatory drugs (NSAIDs) remains under-appreciated. It occurs with greater frequency than the damage caused by these drugs in the upper gastrointestinal tract, but is much more difficult to diagnose and treat. Although the pathogenesis of NSAID enteropathy remains incompletely understood, it is clear that bacteria, bile, and the enterohepatic circulation of NSAIDs are all important factors. However, they are also interrelated with one another. Bacterial enzymes can affect the cytotoxicity of bile and are essential for enterohepatic circulation of NSAIDs. Gram-negative bacteria appear to be particularly important in the pathogenesis of NSAID enteropathy, possibly through release of endotoxin. Inhibitors of gastric acid secretion significantly aggravate NSAID enteropathy, and this effect is due to significant changes in the intestinal microbiome. Treatment with antibiotics can, in some circumstances, reduce the severity of NSAID enteropathy, but published results are inconsistent. Specific antibiotic-induced changes in the microbiota have not been causally linked to prevention of intestinal damage. Treatment with probiotics, particularly Bifidobacterium, Lactobacillus, and Faecalibacteriaum prausnitzii, has shown promising effects in animal models. Our studies suggest that these beneficial effects are due to colonization by the bacteria, rather than to products released by the bacteria.

Longitudinal sampling of the lung microbiota in individuals with cystic fibrosis

Cystic fibrosis (CF) manifests in the lungs resulting in chronic microbial infection. Most morbidity and mortality in CF is due to cycles of pulmonary exacerbations—episodes of acute inflammation in response to the lung microbiome—which are difficult to prevent and treat because their cause is not well understood. We hypothesized that longitudinal analyses of the bacterial component of the CF lung microbiome may elucidate causative agents within this community for pulmonary exacerbations. In this study, 6 participants were sampled thrice-weekly for up to one year. During sampling, sputum, and data (antibiotic usage, spirometry, and symptom scores) were collected. Time points were categorized based on relation to exacerbation as Stable, Intermediate, and Treatment. Retrospectively, a subset of were interrogated via 16S rRNA gene sequencing. When samples were examined categorically, a significant difference between the lung microbiota in Stable, Intermediate, and Treatment samples was observed in a subset of participants. However, when samples were examined longitudinally, no correlations between microbial composition and collected data (antibiotic usage, spirometry, and symptom scores) were observed upon exacerbation onset. In this study, we identified no universal indicator within the lung microbiome of exacerbation onset but instead showed that changes to the CF lung microbiome occur outside of acute pulmonary episodes and are patient-specific.

Streptococcus pneumoniae Colonization Disrupts the Microbial Community within the Upper Respiratory Tract of Aging Mice

ABSTRACT Nasopharyngeal colonization by the Gram-positive bacterium Streptococcus pneumoniae is a prerequisite for pneumonia and invasive pneumococcal diseases. Colonization is asymptomatic, involving dynamic and complex interplay between commensals, the host immune system, and environmental factors. The elderly are at an increased risk of developing pneumonia, which might be due to changes in the respiratory microbiota that would impact bacterial colonization and persistence within this niche. We hypothesized that the composition of the upper respiratory tract (URT) microbiota changes with age and subsequently can contribute to sustained colonization and inefficient clearance of S. pneumoniae. To test this, we used a mouse model of pneumococcal colonization to compare the composition of the URT microbiota in young, middle-aged, and old mice in the naive state and during the course of colonization using nasal pharyngeal washes. Sequencing of variable region 3 (V3) of the 16S rRNA gene was used to identify changes occurring with age and throughout the course of S. pneumoniae colonization. We discovered that age affects the composition of the URT microbiota and that colonization with S. pneumoniae is more disruptive of preexisting communities in older mice. We have further shown that host-pathogen interactions following S. pneumoniae colonization can impact the populations of resident microbes, including Staphylococcus and Haemophilus. Together, our findings indicate alterations to the URT microbiota could be detrimental to the elderly, resulting in increased colonization of S. pneumoniae and decreased efficiency in its clearance.

Non-digestible oligosaccharides directly regulate host kinome to modulate host inflammatory responses without alterations in the gut microbiota

BackgroundPrebiotics are non-digestible food ingredients that enhance the growth of certain microbes within the gut microbiota. Prebiotic consumption generates immune-modulatory effects that are traditionally thought to reflect microbial interactions within the gut. However, recent evidence suggests they may also impart direct microbe-independent effects on the host, though the mechanisms of which are currently unclear.MethodsKinome arrays were used to profile the host intestinal signaling responses to prebiotic exposures in the absence of microbes. Identified pathways were functionally validated in Caco-2Bbe1 intestinal cell line and in vivo model of murine endotoxemia.ResultsWe found that prebiotics directly regulate host mucosal signaling to alter response to bacterial infection. Intestinal epithelial cells (IECs) exposed to prebiotics are hyporesponsive to pathogen-induced mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activations, and have a kinome profile distinct from non-treated cells pertaining to multiple innate immune signaling pathways. Consistent with this finding, mice orally gavaged with prebiotics showed dampened inflammatory response to lipopolysaccharide (LPS) without alterations in the gut microbiota.ConclusionsThese findings provide molecular mechanisms of direct host-prebiotic interactions to support prebiotics as potent modulators of host inflammation.

Assessment of the Microbial Constituents of the Home Environment of Individuals with Cystic Fibrosis (CF) and Their Association with Lower Airways Infections

Introduction Cystic fibrosis (CF) airways are colonized by a polymicrobial community of organisms, termed the CF microbiota. We sought to define the microbial constituents of the home environment of individuals with CF and determine if it may serve as a latent reservoir for infection. Methods Six patients with newly identified CF pathogens were included. An investigator collected repeat sputum and multiple environmental samples from their homes. Bacteria were cultured under both aerobic and anaerobic conditions. Morphologically distinct colonies were selected, purified and identified to the genus and species level through 16S rRNA gene sequencing. When concordant organisms were identified in sputum and environment, pulsed-field gel electrophoresis (PFGE) was performed to determine relatedness. Culture-independent bacterial profiling of each sample was carried out by Illumina sequencing of the V3 region of the 16s RNA gene. Results New respiratory pathogens prompting investigation included: Mycobacterium abscessus(2), Stenotrophomonas maltophilia(3), Pseudomonas aeruginosa(3), Pseudomonas fluorescens(1), Nocardia spp.(1), and Achromobacter xylosoxidans(1). A median 25 organisms/patient were cultured from sputum. A median 125 organisms/home were cultured from environmental sites. Several organisms commonly found in the CF lung microbiome were identified within the home environments of these patients. Concordant species included members of the following genera: Brevibacterium(1), Microbacterium(1), Staphylococcus(3), Stenotrophomonas(2), Streptococcus(2), Sphingomonas(1), and Pseudomonas(4). PFGE confirmed related strains (one episode each of Sphinogomonas and P. aeruginosa) from the environment and airways were identified in two patients. Culture-independent assessment confirmed that many organisms were not identified using culture-dependent techniques. Conclusions Members of the CF microbiota can be found as constituents of the home environment in individuals with CF. While the majority of isolates from the home environment were not genetically related to those isolated from the lower airways of individuals with CF suggesting alternate sources of infection were more common, a few genetically related isolates were indeed identified. As such, the home environment may rarely serve as either the source of infection or a persistent reservoir for re-infection after clearance.

Crystallization and preliminary X-ray diffraction studies of glycerol 3-phosphate cytidylyltransferase from Staphylococcus aureus

Glycerol 3-phosphate cytidylyltransferase from Staphylococcus aureus (TarD(Sa)) has been expressed in Escherichia coli, purified to homogeneity and crystallized. The strategy used for determining crystallization conditions employed hanging-drop sparse-matrix screens and required a combination of three different optimization approaches. Specifically, the presence or absence of cofactors needed to be surveyed, the effects of small-molecule additives required exploration and the rate of vapour-diffusion had to be varied in order to obtain crystals of TarD(Sa) suitable for diffraction studies. Crystals thus obtained belong to the space group P3(1)21, with unit-cell parameters a = b = 92.2, c = 156.1 A, and contain four TarD(Sa) molecules per asymmetric unit. The resolution limit observed for these crystals using synchrotron radiation is 3.0 A.

The Nasopharyngeal Microbiota of Children with Respiratory Infections in Botswana.

Background: Nearly half of child pneumonia deaths occur in sub-Saharan Africa. Microbial communities in the nasopharynx are a reservoir for pneumonia pathogens and remain poorly described in African children. Methods: Nasopharyngeal swabs were collected from children with pneumonia (N = 204), children with upper respiratory infection symptoms (N = 55) and healthy children (N = 60) in Botswana between April 2012 and April 2014. We sequenced the V3 region of the bacterial 16S ribosomal RNA gene and used partitioning around medoids to cluster samples into microbiota biotypes. We then used multivariable logistic regression to examine whether microbiota biotypes were associated with pneumonia and upper respiratory infection symptoms. Results: Mean ages of children with pneumonia, children with upper respiratory infection symptoms and healthy children were 8.2, 11.4 and 8.0 months, respectively. Clustering of nasopharyngeal microbiota identified 5 distinct biotypes: Corynebacterium/Dolosigranulum-dominant (23%), Haemophilus-dominant (11%), Moraxella-dominant (24%), Staphylococcus-dominant (13%) and Streptococcus-dominant (28%). The Haemophilus-dominant [odds ratio (OR): 13.55; 95% confidence interval (CI): 2.10–87.26], the Staphylococcus-dominant (OR: 8.27; 95% CI: 2.13–32.14) and the Streptococcus-dominant (OR: 39.97; 95% CI: 6.63–241.00) biotypes were associated with pneumonia. The Moraxella-dominant (OR: 3.71; 95% CI: 1.09–12.64) and Streptococcus-dominant (OR: 12.26; 95% CI: 1.81–83.06) biotypes were associated with upper respiratory infection symptoms. In children with pneumonia, HIV infection was associated with a lower relative abundance of Dolosigranulum (P = 0.03). Conclusions: Pneumonia and upper respiratory infection symptoms are associated with distinct nasopharyngeal microbiota biotypes in African children. A lower abundance of the commensal genus Dolosigranulum may contribute to the higher pneumonia risk of HIV-infected children.