Exposure to Parasitic Protists and Helminths Changes the Intestinal Community Structure of Bacterial Communities in a Cohort of Mother-Child Binomials from a Semirural Setting in Mexico.

An estimated 3.5 billion people are colonized by intestinal parasites worldwide. Intestinal parasitic eukaryotes interact not only with the host but also with the intestinal microbiota. In this work, we studied the relationship between the presence of multiple enteric parasites and the community structures of gut bacteria and eukaryotes in an asymptomatic mother-child cohort from a semirural community in Mexico. Fecal samples were collected from 46 mothers and their respective children, with ages ranging from 2 to 20 months. Mothers and infants were found to be multiparasitized by Blastocystis hominis, Entamoeba dispar, Endolimax nana, Chilomastix mesnili, Iodamoeba butshlii, Entamoeba coli, Hymenolepis nana, and Ascaris lumbricoides. Sequencing of bacterial 16S rRNA and eukaryotic 18S rRNA genes showed a significant effect of parasite exposure on bacterial beta-diversity, which explained between 5.2% and 15.0% of the variation of the bacterial community structure in the cohort. Additionally, exposure to parasites was associated with significant changes in the relative abundances of multiple bacterial taxa, characterized by an increase in Clostridiales and decreases in Actinobacteria and Bacteroidales. Parasite exposure was not associated with changes in intestinal eukaryote relative abundances. However, we found several significant positive correlations between intestinal bacteria and eukaryotes, including Oscillospira with Entamoeba coli and Prevotella stercorea with Entamoeba hartmanni, as well as the co-occurrence of the fungus Candida with Bacteroides and Actinomyces, Bifidobacterium, and Prevotella copri and the fungus Pichia with Oscillospira. The parasitic exposure-associated changes in the bacterial community structure suggest effects on microbial metabolic routes, host nutrient uptake abilities, and intestinal immunity regulation in host-parasite interactions. IMPORTANCE The impact of intestinal eukaryotes on the prokaryotic microbiome composition of asymptomatic carriers has not been extensively explored, especially in infants and mothers with multiple parasitic infections. In this work, we studied the relationship between protist and helminth parasite colonization and the intestinal microbiota structure in an asymptomatic population of mother-child binomials from a semirural community in Mexico. We found that the presence of parasitic eukaryotes correlated with changes in the bacterial gut community structure in the intestinal microbiota in an age-dependent way. Parasitic infection was associated with an increase in the relative abundance of the class Clostridia and decreases of Actinobacteria and Bacteroidia. Parasitic infection was not associated with changes in the eukaryote community structure. However, we observed strong positive correlations between bacterial and other eukaryote taxa, identifying novel relationships between prokaryotes and fungi reflecting interkingdom interactions within the human intestine.

Shifts in Lachnospira and Clostridium sp. in the 3-month stool microbiome are associated with preschool age asthma.

Asthma is a chronic disease of the airways affecting one in ten children in Westernized countries. Recently, our group showed that specific bacterial genera in early life are associated with atopy and wheezing in 1-year-old children. However, little is known about the link between the early life gut microbiome and the diagnosis of asthma in preschool age children. To determine the role of the gut microbiota in preschool age asthma, children up to 4 years of age enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) study were classified as asthmatic (n=39) or matched healthy controls (n=37). 16S rRNA sequencing and quantitative PCR (qPCR) were used to analyse the composition of the 3-month and 1-year gut microbiome of these children. At 3 months the abundance of the genus, Lachnospira (L), was decreased (P=0.008), whereas the abundance of the species, Clostridium neonatale (C), was increased (P=0.07) in asthmatics. Quartile analysis of stool composition at 3-months revealed a negative association between the ratio of these two bacteria (L/C) and asthma risk by 4 years of age [quartile 1: odds ratio (OR)=15, P=0.02, CI (confidence interval)= 1.8-124.7; quartile 2: OR=1.0, ns; quartile 3: OR=0.37, ns]. We conclude that opposing shifts in the relative abundances of Lachnospira and C. neonatale in the first 3 months of life are associated with preschool age asthma, and that the L/C ratio may serve as a potential early life biomarker to predict asthma development.

Early infancy microbial and metabolic alterations affect risk of childhood asthma.

Asthma is the most prevalent pediatric chronic disease and affects more than 300 million people worldwide. Recent evidence in mice has identified a "critical window" early in life where gut microbial changes (dysbiosis) are most influential in experimental asthma. However, current research has yet to establish whether these changes precede or are involved in human asthma. We compared the gut microbiota of 319 subjects enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) Study, and show that infants at risk of asthma exhibited transient gut microbial dysbiosis during the first 100 days of life. The relative abundance of the bacterial genera Lachnospira, Veillonella, Faecalibacterium, and Rothia was significantly decreased in children at risk of asthma. This reduction in bacterial taxa was accompanied by reduced levels of fecal acetate and dysregulation of enterohepatic metabolites. Inoculation of germ-free mice with these four bacterial taxa ameliorated airway inflammation in their adult progeny, demonstrating a causal role of these bacterial taxa in averting asthma development. These results enhance the potential for future microbe-based diagnostics and therapies, potentially in the form of probiotics, to prevent the development of asthma and other related allergic diseases in children.

Perinatal antibiotic-induced shifts in gut microbiota have differential effects on inflammatory lung diseases.

BACKGROUND: Resident gut microbiota are now recognized as potent modifiers of host immune responses in various scenarios. Recently, we demonstrated that perinatal exposure to vancomycin, but not streptomycin, profoundly alters gut microbiota and enhances susceptibility to a TH2 model of allergic asthma. OBJECTIVE: Here we sought to further clarify the etiology of these changes by determining whether perinatal antibiotic treatment has a similar effect on the TH1/TH17-mediated lung disease, hypersensitivity pneumonitis. METHODS: Hypersensitivity pneumonitis was induced in C57BL/6 wild-type or recombination-activating gene 1-deficient mice treated perinatally with vancomycin or streptomycin by repeated intranasal administration of Saccharopolyspora rectivirgula antigen. Disease severity was assessed by measuring lung inflammation, pathology, cytokine responses, and serum antibodies. Microbial community analyses were performed on stool samples via 16S ribosomal RNA pyrosequencing and correlations between disease severity and specific bacterial taxa were identified. RESULTS: Surprisingly, in contrast to our findings in an allergic asthma model, we found that the severity of hypersensitivity pneumonitis was unaffected by vancomycin, but increased dramatically after streptomycin treatment. This likely reflects an effect on the adaptive, rather than innate, immune response because the effects of streptomycin were not observed during the early phases of disease and were abrogated in recombination-activating gene 1-deficient mice. Interestingly, Bacteroidetes dominated the intestinal microbiota of streptomycin-treated animals, while vancomycin promoted the expansion of the Firmicutes. CONCLUSIONS: Perinatal antibiotics exert highly selective effects on resident gut flora, which, in turn, lead to very specific alterations in susceptibility to TH2- or TH1/TH17-driven lung inflammatory disease.

Discovery and optimization of a new class of pyruvate kinase inhibitors as potential therapeutics for the treatment of methicillin-resistant Staphylococcus aureus infections.

A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is not only critical for bacterial survival which would make it a target for development of novel antibiotics, but it is reported to be one of the most highly connected 'hub proteins' in MRSA, and thus should be very sensitive to mutations and making it difficult for the bacteria to develop resistance. From the co-crystal structure of cis-3-4-dihydrohamacanthin B (4) bound to S. aureus PK we were able to identify the pharmacophore needed for activity. Consequently, we prepared simple direct linked bis-indoles such as 10b that have similar anti-MRSA activity as compound 4. Structure-activity relationship (SAR) studies were carried out on 10b and led us to discover more potent compounds such as 10c, 10d, 10k and 10 m with enzyme inhibiting activities in the low nanomolar range that effectively inhibited the bacteria growth in culture with minimum inhibitory concentrations (MIC) for MRSA as low as 0.5 µg/ml. Some potent PK inhibitors, such as 10b, exhibited attenuated antibacterial activity and were found to be substrates for an efflux mechanism in S. aureus. Studies comparing a wild type S. aureus with a construct (S. aureus LAC Δpyk::Erm(R)) that lacks PK activity confirmed that bactericidal activity of 10d was PK-dependant.

Perinatal antibiotic treatment affects murine microbiota, immune responses and allergic asthma.

There is convincing evidence from recent human and animal studies that suggests the intestinal microbiota plays an important role in regulating immune responses associated with the development of allergic asthma, particularly during early infancy. Although identifying the mechanistic link between host-microbe interactions in the gut and lung mucosal tissues has proved challenging, several very recent studies are now providing significant insights. We have shown that administering vancomycin to mice early in life shifts resident gut flora and enhances future susceptibility to allergic asthma. This effect was not observed in mice given another antibiotic, streptomycin, nor when either antibiotic was administered to adult mice. In this addendum, we further analyze the link between early life administration of vancomycin and future susceptibility to asthma and describe how specific immune cell populations, which have been implicated in other asthma-related microbiota studies, are affected. We propose that shifts in gut microbiota exacerbate asthma-related immune responses when they occur shortly after birth and before weaning (perinatal period), and suggest that these effects may be mediated, at least in the case of vancomycin, by elevated serum IgE and reduced regulatory T cell populations.

Manipulation of innate immunity by a bacterial secreted peptide: lantibiotic nisin Z is selectively immunomodulatory.

Innate immunity is triggered by a variety of bacterial molecules, resulting in both protective and potentially harmful pro-inflammatory responses. Further, innate immunity also provides a mechanism for the maintenance of homeostasis between the host immune system and symbiotic or non-pathogenic microorganisms. However, the bacterial factors that mediate these protective effects have been incompletely defined. Here, it was demonstrated that the lantiobiotic nisin Z is able to modulate host immune responses and mediate protective host immunity. Nisin Z induced the secretion of the chemokines MCP-1, IL-8 and Gro-α, and significantly reduced TNF-α induction in response to bacterial LPS in human PBMC. The results correlated with the ability of nisin Z to confer protection against both the Gram-positive organism Staphylococcus aureus, and the Gram-negatives Salmonella enterica sv. Typhimurium and Escherichia coli in murine challenge models. Mechanistic studies revealed that nisin Z modulates host immunity through similar mechanisms as natural host defense peptides, engaging multiple signal transduction pathways and growth factor receptors. The results presented herein demonstrate that, in addition to nisin Z, other bacterial cationic peptides and, in particular, the lantibiotics, could represent a new class of secreted bacterial molecule with immunomodulatory activities.

Optimization and structure-activity relationships of a series of potent inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as novel antimicrobial agents.

A novel series of hydrazones were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as one of the most highly connected 'hub proteins' in MRSA. PK has been shown to be critical for bacterial survival which makes it a potential target for development of novel antibiotics and the high degree of connectivity implies it should be very sensitive to mutations and thus less able to develop resistance. PK is not unique to bacteria and thus a critical requirement for such a PK inhibitor would be that it does not inhibit the homologous human enzyme(s) at therapeutic concentrations. Several MRSA PK inhibitors (including 8d) were identified using in silico screening combined with enzyme assays and were found to be selective for bacterial enzyme compared to four human PK isoforms (M1, M2, R and L). However these lead compounds did not show significant inhibitory activity for MRSA growth presumably due to poor bacterial cell penetration. Structure-activity relationship (SAR) studies were carried out on 8d and led us to discover more potent compounds with enzyme inhibiting activities in the low nanomolar range and some were found to effectively inhibit bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1 µg/mL. These inhibitors bind in two elongated flat clefts found at the minor interfaces in the homo-tetrameric enzyme complex and the observed SAR is in keeping with the size and electronic constraints of these binding sites. Access to the corresponding sites in the human enzyme is blocked.

Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma.

Allergic asthma rates have increased steadily in developed countries, arguing for an environmental aetiology. To assess the influence of gut microbiota on experimental murine allergic asthma, we treated neonatal mice with clinical doses of two widely used antibiotics--streptomycin and vancomycin--and evaluated resulting shifts in resident flora and subsequent susceptibility to allergic asthma. Streptomycin treatment had little effect on the microbiota and on disease, whereas vancomycin reduced microbial diversity, shifted the composition of the bacterial population and enhanced disease severity. Neither antibiotic had a significant effect when administered to adult mice. Consistent with the 'hygiene hypothesis', our data support a neonatal, microbiota-driven, specific increase in susceptibility to experimental murine allergic asthma.

Identification of pyruvate kinase in methicillin-resistant Staphylococcus aureus as a novel antimicrobial drug target.

Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Using the architecture of the MRSA interactome, we identified pyruvate kinase (PK) as a potential novel drug target based upon it being a highly connected, essential hub in the MRSA interactome. Structural modeling, including X-ray crystallography, revealed discrete features of PK in MRSA, which appeared suitable for the selective targeting of the bacterial enzyme. In silico library screening combined with functional enzymatic assays identified an acyl hydrazone-based compound (IS-130) as a potent MRSA PK inhibitor (50% inhibitory concentration [IC50] of 0.1 µM) with >1,000-fold selectivity over human PK isoforms. Medicinal chemistry around the IS-130 scaffold identified analogs that more potently and selectively inhibited MRSA PK enzymatic activity and S. aureus growth in vitro (MIC of 1 to 5 µg/ml). These novel anti-PK compounds were found to possess antistaphylococcal activity, including both MRSA and multidrug-resistant S. aureus (MDRSA) strains. These compounds also exhibited exceptional antibacterial activities against other Gram-positive genera, including enterococci and streptococci. PK lead compounds were found to be noncompetitive inhibitors and were bactericidal. In addition, mutants with significant increases in MICs were not isolated after 25 bacterial passages in culture, indicating that resistance may be slow to emerge. These findings validate the principles of network science as a powerful approach to identify novel antibacterial drug targets. They also provide a proof of principle, based upon PK in MRSA, for a research platform aimed at discovering and optimizing selective inhibitors of novel bacterial targets where human orthologs exist, as leads for anti-infective drug development.

Salmonella infection of gallbladder epithelial cells drives local inflammation and injury in a model of acute typhoid fever.

The gallbladder is often colonized by Salmonella during typhoid fever, yet little is known about bacterial pathogenesis in this organ. With use of a mouse model of acute typhoid fever, we demonstrate that Salmonella infect gallbladder epithelial cells in vivo. Bacteria in the gallbladder showed a unique behavior as they replicated within gallbladder epithelial cells and remained confined to those cells without translocating to the mucosa. Infected gallbladders showed histopathological damage characterized by destruction of the epithelium and massive infiltration of neutrophils, accompanied by a local increase of proinflammatory cytokines. Damage was determined by the ability of Salmonella to invade gallbladder epithelial cells and was independent of high numbers of replication-competent, although invasion-deficient, bacteria in the lumen. Our results establish gallbladder epithelial cells as a novel niche for in vivo replication of Salmonella and reveal the involvement of these cells in the pathogenesis of Salmonella in the gallbladder during the course of acute typhoid fever.

Sequestosome-1/p62 is the key intracellular target of innate defense regulator peptide.

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor alpha-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor alpha-induced p62-protein kinase zeta complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein beta activity, whereas NF-kappaB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.

Role of the hypoglossal nerve in equine nasopharyngeal stability.

The equine upper airway is highly adapted to provide the extremely high oxygen demand associated with strenuous aerobic exercise in this species. The tongue musculature, innervated by the hypoglossal nerve, plays an important role in airway stability in humans who also have a highly adapted upper airway to allow speech. The role of the hypoglossal nerve in stabilizing the equine upper airway has not been established. Isolated tongues from eight mature horses were dissected to determine the distal anatomy and branching of the equine hypoglossal nerve. Using this information, a peripheral nerve location technique was used to perform bilateral block of the common trunk of the hypoglossal nerve in 10 horses. Each horse was subjected to two trials with bilateral hypoglossal nerve block and two control trials (unblocked). Upper airway stability at exercise was determined using videoendoscopy and measurement of tracheal and pharyngeal pressure. Three main nerve branches were identified, medial and lateral branches and a discrete branch that innervated the geniohyoid muscle alone. Bilateral hypoglossal block induced nasopharyngeal instability in 10/19 trials, and none of the control trials (0/18) resulted in instability (P<0.001). Mean treadmill speed (+/-SD) at the onset of instability was 10.8+/-2.5 m/s. Following its onset, nasopharyngeal instability persisted until the end of the treadmill test. This instability, induced by hypoglossal nerve block, produced an expiratory obstruction similar to that seen in a naturally occurring equine disease (dorsal displacement of the soft palate, DDSP) with reduced inspiratory and expiratory pharyngeal pressure and increased expiratory tracheal pressure. These data suggest that stability of the equine upper airway at exercise may be mediated through the hypoglossal nerve. Naturally occurring DDSP in the horse shares a number of anatomic similarities with obstructive sleep apnea. Study of species with extreme respiratory adaptation, such as the horse, may provide insight into respiratory functioning in humans.

Mycobacterium tuberculosis Cpn60.2 and DnaK are located on the bacterial surface, where Cpn60.2 facilitates efficient bacterial association with macrophages.

Mycobacterium tuberculosis, the causative agent of tuberculosis, initially contacts host cells with elements of its outer cell wall, or capsule. We have shown that capsular material from the surface of M. tuberculosis competitively inhibits the nonopsonic binding of whole M. tuberculosis bacilli to macrophages in a dose-dependent manner that is not acting through a global inhibition of macrophage binding. We have further demonstrated that isolated M. tuberculosis capsular proteins mediate a major part of this inhibition. Two-dimensional polyacrylamide gel electrophoresis analysis of the capsular proteins showed the presence of a wide variety of protein species, including proportionately high levels of the Cpn60.2 (Hsp65, GroEL2) and DnaK (Hsp70) molecular chaperones. Both of these proteins were subsequently detected on the bacterial surface. To determine whether these molecular chaperones play a role in bacterial binding, recombinant Cpn60.2 and DnaK were tested for their ability to inhibit the association of M. tuberculosis bacilli with macrophages. We found that recombinant Cpn60.2 can inhibit approximately 57% of bacterial association with macrophages, while DnaK was not inhibitory at comparable concentrations. Additionally, when polyclonal F(ab')(2) fragments of anti-Cpn60.2 and anti-DnaK were used to mask the surface presentation of these molecular chaperones, a binding reduction of approximately 34% was seen for anti-Cpn60.2 F(ab')(2), while anti-DnaK F(ab')(2) did not significantly reduce bacterial association with macrophages. Thus, our findings suggest that while M. tuberculosis displays both surface-associated Cpn60.2 and DnaK, only Cpn60.2 demonstrates adhesin functionality with regard to macrophage interaction.

A murine intraperitoneal infection model reveals that host resistance to Campylobacter jejuni is Nramp1 dependent.

We tested the hypothesis that host resistance to Campylobacter jejuni is Nramp1 dependent. Following intraperitoneal (IP) inoculation of Nramp1+/+ and isogenic Nramp1-deficient (Nramp1-/-) mice C. jejuni primarily associated with mac1-positive cells in liver tissue. A significant reduction of C. jejuni was observed in Nramp1+/+ mice 4 days post-infection (PI) (liver) and 8 days PI cecum-colon. In contrast, Nramp1-/- mice showed no significant reduction of C. jejuni and instead had a chronic inflammatory response and significant histopathological lesions 30 days PI. Differential cytokine profiles were observed in C. jejuni infected Nramp1+/+ and Nramp1-/- primary dendritic cells. Taken together these data indicate that Nramp1 is critical for host resistance to C. jejuni.

Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis.

Bacillus Calmette-Guerin (BCG) vaccine has failed to control the global tuberculosis (TB) epidemic, and there is a lack of safe and effective mucosal vaccines capable of potent protection against pulmonary TB. A recombinant replication-deficient adenoviral-based vaccine expressing an immunogenic Mycobacterium tuberculosis Ag Ag85A (AdAg85A) was engineered and evaluated for its potential to be used as a respiratory mucosal TB vaccine in a murine model of pulmonary TB. A single intranasal, but not i.m., immunization with AdAg85A provided potent protection against airway Mycobacterium tuberculosis challenge at an improved level over that by cutaneous BCG vaccination. Systemic priming with an Ag85A DNA vaccine and mucosal boosting with AdAg85A conferred a further enhanced immune protection which was remarkably better than BCG vaccination. Such superior protection triggered by AdAg85 mucosal immunization was correlated with much greater retention of Ag-specific T cells, particularly CD4 T cells, in the lung and was shown to be mediated by both CD4 and CD8 T cells. Thus, adenoviral TB vaccine represents a promising novel vaccine platform capable of potent mucosal immune protection against TB. Our study also lends strong evidence that respiratory mucosal vaccination is critically advantageous over systemic routes of vaccination against TB.

The glycan-rich outer layer of the cell wall of Mycobacterium tuberculosis acts as an antiphagocytic capsule limiting the association of the bacterium with macrophages.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that infects macrophages and other host cells. We show that sonication of M. tuberculosis results in the removal of material from the surface capsule-like layer of the bacteria, resulting in an enhanced propensity of the bacteria to bind to macrophages. This effect is observed with disparate murine and human macrophage populations though, interestingly, not with freshly explanted alveolar macrophages. Enhanced binding to macrophages following sonication is significantly greater within members of the M. tuberculosis family (pathogens) than within the Mycobacterium avium complex (opportunistic pathogens) or for Mycobacterium smegmatis (saprophyte). Sonication does not affect the viability or the surface hydrophobicity of M. tuberculosis but does result in changes in surface charge and in the binding of mannose-specific lectins to the bacterial surface. The increased binding of sonicated M. tuberculosis was not mediated through complement receptor 3. These results provide evidence that the surface capsule on members of the M. tuberculosis family may be an important virulence factor involved in the survival of M. tuberculosis in the mammalian host. They also question the view that M. tuberculosis is readily ingested by any macrophage it encounters and support the contention that M. tuberculosis, like many other microbial pathogens, has an antiphagocytic capsule that limits and controls the interaction of the bacterium with macrophages.