Showing papers in "Mbio in 2011"

Phylogenetic Molecular Ecological Network of Soil Microbial Communities in Response to Elevated CO2

Abstract: Understanding the interactions among different species and their responses to environmental changes, such as ele- vated atmospheric concentrations of CO2, is a central goal in ecology but is poorly understood in microbial ecology. Here we describe a novel random matrix theory (RMT)-based conceptual framework to discern phylogenetic molecular ecological net- works using metagenomic sequencing data of 16S rRNA genes from grassland soil microbial communities, which were sampled from a long-term free-air CO2enrichment experimental facility at the Cedar Creek Ecosystem Science Reserve in Minnesota. Our experimental results demonstrated that an RMT-based network approach is very useful in delineating phylogenetic molecu- lar ecological networks of microbial communities based on high-throughput metagenomic sequencing data. The structure of the identified networks under ambient and elevated CO 2levels was substantially different in terms of overall network topology, net- work composition, node overlap, module preservation, module-based higher-order organization, topological roles of individual nodes, and network hubs, suggesting that the network interactions among different phylogenetic groups/populations were markedly changed. Also, the changes in network structure were significantly correlated with soil carbon and nitrogen contents, indicating the potential importance of network interactions in ecosystem functioning. In addition, based on network topology, microbial populations potentially most important to community structure and ecosystem functioning can be discerned. The novel approach described in this study is important not only for research on biodiversity, microbial ecology, and systems micro- biology but also for microbial community studies in human health, global change, and environmental management. IMPORTANCE The interactions among different microbial populations in a community play critical roles in determining ecosys- tem functioning, but very little is known about the network interactions in a microbial community, owing to the lack of appro- priate experimental data and computational analytic tools. High-throughput metagenomic technologies can rapidly produce a massive amount of data, but one of the greatest difficulties is deciding how to extract, analyze, synthesize, and transform such a vast amount of information into biological knowledge. This study provides a novel conceptual framework to identify microbial interactions and key populations based on high-throughput metagenomic sequencing data. This study is among thefirst to doc- ument that the network interactions among different phylogenetic populations in soil microbial communities were substantially changed by a global change such as an elevated CO2level. The framework developed will allow microbiologists to address re- search questions which could not be approached previously, and hence, it could represent a new direction in microbial ecology research.

Potential for Direct Interspecies Electron Transfer in Methanogenic Wastewater Digester Aggregates

Abstract: Mechanisms for electron transfer within microbial aggregates derived from an upflow anaerobic sludge blanket reac- tor converting brewery waste to methane were investigated in order to better understand the function of methanogenic consor- tia. The aggregates were electrically conductive, with conductivities 3-fold higher than the conductivities previously reported for dual-species aggregates of Geobacterspecies in which the two species appeared to exchange electrons via interspecies electron transfer. The temperature dependence response of the aggregate conductance was characteristic of the organic metallic-like con- ductance previously described for the conductive pili of Geobacter sulfurreducens and was inconsistent with electron conduction through minerals. Studies in which aggregates were incubated with high concentrations of potential electron donors demon- strated that the aggregates had no significant capacity for conversion of hydrogen to methane. The aggregates converted formate to methane but at rates too low to account for the rates at which that the aggregates syntrophically metabolized ethanol, an im- portant component of the reactor influent. Geobacterspecies comprised 25% of 16S rRNA gene sequences recovered from the aggregates, suggesting that Geobacterspecies may have contributed to some but probably not all of the aggregate conductivity. Microorganisms most closely related to the acetate-utilizing Methanosaeta concilii accounted for more than 90% of the se- quences that could be assigned to methane producers, consistent with the poor capacity for hydrogen and formate utilization. These results demonstrate for thefirst time that methanogenic wastewater aggregates can be electrically conductive and suggest that direct interspecies electron transfer could be an important mechanism for electron exchange in some methanogenic sys- tems. IMPORTANCE The conversion of waste organic matter to methane is an important bioenergy strategy, and a similar microbial metabolism of complex organic matter in anaerobic soils and sediments plays an important role in the global carbon cycle. Stud- ies with laboratory cultures have demonstrated that hydrogen or formate can serve as an electron shuttle between the microor- ganisms degrading organic compounds and methanogens. However, the importance of hydrogen and formate as intermediates in the conversion of organic matter to methane in natural communities is less clear. The possibility that microorganisms within some natural methanogenic aggregates may directly exchange electrons, rather than producing hydrogen or formate as an inter- mediary electron carrier, is a significant paradigm shift with implications for the modeling and design of anaerobic wastewater reactors and for understanding how methanogenic communities will respond to environmental perturbations.

Colonization-Induced Host-Gut Microbial Metabolic Interaction

Abstract: The gut microbiota enhances the host’s metabolic capacity for processing nutrients and drugs and modulate the activities of multiple pathways in a variety of organ systems. We have probed the systemic metabolic adaptation to gut colonization for 20 days following exposure of axenic mice ( n = 35) to a typical environmental microbial background using high-resolution 1 H nuclear magnetic resonance (NMR) spectroscopy to analyze urine, plasma, liver, kidney, and colon (5 time points) metabolic profiles. Acquisition of the gut microbiota was associated with rapid increase in body weight (4%) over the first 5 days of colonization with parallel changes in multiple pathways in all compartments analyzed. The colonization process stimulated glycogenesis in the liver prior to triggering increases in hepatic triglyceride synthesis. These changes were associated with modifications of hepatic Cyp8b1 expression and the subsequent alteration of bile acid metabolites, including taurocholate and tauromuricholate, which are essential regulators of lipid absorption. Expression and activity of major drug-metabolizing enzymes (Cyp3a11 and Cyp2c29) were also significantly stimulated. Remarkably, statistical modeling of the interactions between hepatic metabolic profiles and microbial composition analyzed by 16S rRNA gene pyrosequencing revealed strong associations of the Coriobacteriaceae family with both the hepatic triglyceride, glucose, and glycogen levels and the metabolism of xenobiotics. These data demonstrate the importance of microbial activity in metabolic phenotype development, indicating that microbiota manipulation is a useful tool for beneficially modulating xenobiotic metabolism and pharmacokinetics in personalized health care. IMPORTANCE Gut bacteria have been associated with various essential biological functions in humans such as energy harvest and regulation of blood pressure. Furthermore, gut microbial colonization occurs after birth in parallel with other critical processes such as immune and cognitive development. Thus, it is essential to understand the bidirectional interaction between the host metabolism and its symbionts. Here, we describe the first evidence of an in vivo association between a family of bacteria and hepatic lipid metabolism. These results provide new insights into the fundamental mechanisms that regulate host-gut microbiota interactions and are thus of wide interest to microbiological, nutrition, metabolic, systems biology, and pharmaceutical research communities. This work will also contribute to developing novel strategies in the alteration of host-gut microbiota relationships which can in turn beneficially modulate the host metabolism.

Characterization and Transcriptome Analysis of Mycobacterium tuberculosis Persisters

Abstract: Tuberculosis continues to be a major public health problem in many parts of the world. Significant obstacles in controlling the epidemic are the length of treatment and the large reservoir of latently infected people. Bacteria form dormant, drug-tolerant persister cells, which may be responsible for the difficulty in treating both acute and latent infections. We find that in Mycobacterium tuberculosis, low numbers of drug-tolerant persisters are present in lag and early exponential phases, increasing sharply at late exponential and stationary phases to make up ~1% of the population. This suggests that persister formation is governed by both stochastic and deterministic mechanisms. In order to isolate persisters, an exponentially growing population was treated with d-cycloserine, and cells surviving lysis were collected by centrifugation. A transcriptome of persisters was obtained by using hybridization to an Affymetrix array. The transcriptome shows downregulation of metabolic and biosynthetic pathways, consistent with a certain degree of dormancy. A set of genes was upregulated in persisters, and these are likely involved in persister formation and maintenance. A comparison of the persister transcriptome with transcriptomes obtained for several in vitro dormancy models identified a small number of genes upregulated in all cases, which may represent a core dormancy response.

Antimicrobial Actions of Reactive Oxygen Species

Abstract: Everything should be as simple as it can be, but not simpler. —Attributed to Albert Einstein (1) IMPORTANCE Reactive oxygen species (ROS) are produced by host phagocytes and exert antimicrobial actions against a broad range of pathogens. The observable antimicrobial actions of ROS are highly dependent on experimental conditions. This perspective reviews recent controversies regarding ROS in Salmonella -phagocyte interactions and attempts to reconcile conflicting observations from different laboratories.

Population Genetics of Vibrio cholerae from Nepal in 2010: Evidence on the Origin of the Haitian Outbreak

Abstract: Cholera continues to be an important cause of human infections, and outbreaks are often observed after natural disasters, such as the one following the 2010 earthquake in Haiti. Once the cholera outbreak was confirmed, rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. We used whole-genome sequence typing (WGST), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing to characterize 24 recent Vibrio cholerae isolates from Nepal and evaluate the suggested epidemiological link with the Haitian outbreak. The isolates were obtained from 30 July to 1 November 2010 from five different districts in Nepal. We compared the 24 genomes to 10 previously sequenced V. cholerae isolates, including 3 from the Haitian outbreak (began July 2010). Antimicrobial susceptibility and PFGE patterns were consistent with an epidemiological link between the isolates from Nepal and Haiti. WGST showed that all 24 V. cholerae isolates from Nepal belonged to a single monophyletic group that also contained isolates from Bangladesh and Haiti. The Nepalese isolates were divided into four closely related clusters. One cluster contained three Nepalese isolates and three Haitian isolates that were almost identical, with only 1- or 2-bp differences. Results in this study are consistent with Nepal as the origin of the Haitian outbreak. This highlights how rapidly infectious diseases might be transmitted globally through international travel and how public health officials need advanced molecular tools along with standard epidemiological analyses to quickly determine the sources of outbreaks.

The Fungal Pathogen Candida albicans Autoinduces Hyphal Morphogenesis by Raising Extracellular pH

Abstract: pH homeostasis is critical for all organisms; in the fungal pathogen Candida albicans, pH adaptation is critical for virulence in distinct host niches. We demonstrate that beyond adaptation, C. albicans actively neutralizes the environment from either acidic or alkaline pHs. Under acidic conditions, this species can raise the pH from 4 to >7 in less than 12 h, resulting in autoinduction of the yeast-hyphal transition, a critical virulence trait. Extracellular alkalinization has been reported to occur in several fungal species, but under the specific conditions that we describe, the phenomenon is more rapid than previously observed. Alkalinization is linked to carbon deprivation, as it occurs in glucose-poor media and requires exogenous amino acids. These conditions are similar to those predicted to exist inside phagocytic cells, and we find a strong correlation between the use of amino acids as a cellular carbon source and the degree of alkalinization. Genetic and genomic approaches indicate an emphasis on amino acid uptake and catabolism in alkalinizing cells. Mutations in four genes, STP2 , a transcription factor regulating amino acid permeases, ACH1 (acetyl-coenzyme A [acetyl-CoA] hydrolase), DUR1 , 2 (urea amidolyase), and ATO5 , a putative ammonia transporter, abolish or delay neutralization. The pH changes are the result of the extrusion of ammonia, as observed in other fungi. We propose that nutrient-deprived C. albicans cells catabolize amino acids as a carbon source, excreting the amino nitrogen as ammonia to raise environmental pH and stimulate morphogenesis, thus directly contributing to pathogenesis. IMPORTANCE Candida albicans is the most important fungal pathogen of humans, causing disease at multiple body sites. The ability to switch between multiple morphologies, including a rounded yeast cell and an elongated hyphal cell, is a key virulence trait in this species, as this reversible switch is thought to promote dissemination and tissue invasion in the host. We report here that C. albicans can actively alter the pH of its environment and induce its switch to the hyphal form. The change in pH is caused by the release of ammonia from the cells produced during the breakdown of amino acids. This phenomenon is unprecedented in a human pathogen and may substantially impact host physiology by linking morphogenesis, pH adaptation, carbon metabolism, and interactions with host cells, all of which are critical for the ability of C. albicans to cause disease.

Raw Sewage Harbors Diverse Viral Populations

Abstract: At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included vi- ruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA (ssRNA()), and dsRNA ge- nomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity. IMPORTANCE At this time, virology is focused on the study of a relatively small number of viral species. Specific viruses are stud- ied either because they are easily propagated in the laboratory or because they are associated with disease. The lack of knowledge of the size and characteristics of the viral universe and the diversity of viral genomes is a roadblock to understanding important issues, such as the origin of emerging pathogens and the extent of gene exchange among viruses. Untreated wastewater is an ideal system for assessing viral diversity because virion populations from large numbers of individuals are deposited and because raw sewage itself provides a rich environment for the growth of diverse host species and thus their viruses. These studies suggest that the viral universe is far more vast and diverse than previously suspected.

Impact of pneumococcal conjugate vaccination of infants on pneumonia and influenza hospitalization and mortality in all age groups in the United States.

Abstract: A seven-valent pneumococcal conjugate vaccine (PCV7) introduced in the United States in 2000 has been shown to reduce invasive pneumococcal disease (IPD) in both vaccinated children and adults through induction of herd immunity. We assessed the impact of infant immunization on pneumococcal pneumonia hospitalizations and mortality in all age groups using Health Care Utilization Project State Inpatient Databases (SID) for 1996 to 2006 from 10 states; SID contain 100% samples of ICD9-coded hospitalization data for the selected states. Compared to a 1996-1997 through 1998-1999 baseline, by the 2005-2006 season, both IPD and pneumococcal pneumonia hospitalizations and deaths had decreased substantially in all age groups, including a 47% (95% confidence interval [CI], 38 to 54%) reduction in nonbacteremic pneumococcal pneumonia (ICD9 code 481 with no codes indicating IPD) in infants <2 years old and a 54% reduction (CI, 53 to 56%) in adults ≥65 years of age. A model developed to calculate the total burden of pneumococcal pneumonia prevented by infant PCV7 vaccination in the United States from 2000 to 2006 estimated a reduction of 788,838 (CI, 695,406 to 875,476) hospitalizations for pneumococcal pneumonia. Ninety percent of the reduction in model-attributed pneumococcal pneumonia hospitalizations occurred through herd immunity among adults 18 years old and older; similar proportions were found in pneumococcal disease mortality prevented by the vaccine. In the first seasons after PCV introduction, when there were substantial state differences in coverage among <5-year-olds, states with greater coverage had significantly fewer influenza-associated pneumonia hospitalizations among children, suggesting that PCV7 use also reduces influenza-attributable pneumonia hospitalizations.

Genome-Scale Identification of Resistance Functions in Pseudomonas aeruginosa Using Tn-seq

Abstract: We describe a deep-sequencing procedure for tracking large numbers of transposon mutants of Pseudomonas aeruginosa. The procedure employs a new Tn-seq methodology based on the generation and amplification of single-strand circles carrying transposon junction sequences (the Tn-seq circle method), a method which can be used with virtually any transposon. The procedure reliably identified more than 100,000 transposon insertions in a single experiment, providing near-saturation coverage of the genome. To test the effectiveness of the procedure for mutant identification, we screened for mutations reducing intrinsic resistance to the aminoglycoside antibiotic tobramycin. Intrinsic tobramycin resistance had been previously analyzed at genome scale using mutant-by-mutant screening and thus provided a benchmark for evaluating the new method. The new Tn-seq procedure identified 117 tobramycin resistance genes, the majority of which were then verified with individual mutants. The group of genes with the strongest mutant phenotypes included nearly all (13 of 14) of those with strong mutant phenotypes identified in the previous screening, as well as a nearly equal number of new genes. The results thus show the effectiveness of the Tn-seq method in defining the genetic basis of a complex resistance trait of P. aeruginosa and indicate that it can be used to analyze a variety of growth-related processes. IMPORTANCE Research progress in microbiology is technology limited in the sense that the analytical methods available dictate how questions are experimentally addressed and, to some extent, what questions are asked. This report describes a new transposon tracking procedure for defining the genetic basis of growth-related processes in Pseudomonas aeruginosa, an important bacterial pathogen. The method employs next-generation sequencing to monitor the makeup of mutant populations (Tn-seq) and has several potential advantages over other Tn-seq methodologies. The new method was validated through the analysis of a clinically relevant antibiotic resistance trait.

Dot/Icm Type IVB Secretion System Requirements for Coxiella burnetii Growth in Human Macrophages

Abstract: Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, within a phagolysosome-like parasitophorous vacuole (PV) in mononuclear phagocytes. C. burnetii modulates PV biogenesis and other host cell functions, such as apoptotic signaling, presumably via the activity of proteins delivered to the host cytosol by a Dot/Icm type IVB secretion system (T4BSS). In this study, we utilized a C. burnetii strain carrying IcmD inactivated by the Himar1 transposon to investigate the requirements for Dot/Icm function in C. burnetii parasitism of human THP-1 macrophage-like cells. The icmD::Tn mutant failed to secrete characterized T4BSS substrates, a defect that correlated with deficient replication, PV development, and apoptosis protection. Restoration of type IVB secretion and intracellular growth of the icmD::Tn mutant required complementation with icmD, -J, and -B, indicating a polar effect of the transposon insertion on downstream dot/icm genes. Induction of icmDJB expression at 1 day postinfection resulted in C. burnetii replication and PV generation. Collectively, these data prove that T4BSS function is required for productive infection of human macrophages by C. burnetii. However, illustrating the metabolic flexibility of C. burnetti, the icmD::Tn mutant could replicate intracellularly when sequestered in a PV generated by wild-type bacteria, where Dot/Icm function is provided in trans, and within a phenotypically similar PV generated by the protozoan parasite Leishmania amazonensis, where host cells are devoid of Dot/Icm T4BSS effector proteins.

Antibiotics in Feed Induce Prophages in Swine Fecal Microbiomes

Abstract: Antibiotics are a cost-effective tool for improving feed efficiency and preventing disease in agricultural animals, but the full scope of their collateral effects is not understood. Antibiotics have been shown to mediate gene transfer by inducing prophages in certain bacterial strains; therefore, one collateral effect could be prophage induction in the gut microbiome at large. Here we used metagenomics to evaluate the effect of two antibiotics in feed (carbadox and ASP250 [chlortetracycline, sulfamethazine, and penicillin]) on swine intestinal phage metagenomes (viromes). We also monitored the bacterial communities using 16S rRNA gene sequencing. ASP250, but not carbadox, caused significant population shifts in both the phage and bacterial communities. Antibiotic resistance genes, such as multidrug resistance efflux pumps, were identified in the viromes, but in-feed antibiotics caused no significant changes in their abundance. The abundance of phage integrase-encoding genes was significantly increased in the viromes of medicated swine over that in the viromes of nonmedicated swine, demonstrating the induction of prophages with antibiotic treatment. Phage-bacterium population dynamics were also examined. We observed a decrease in the relative abundance of Streptococcus bacteria (prey) when Streptococcus phages (predators) were abundant, supporting the “kill-the-winner” ecological model of population dynamics in the swine fecal microbiome. The data show that gut ecosystem dynamics are influenced by phages and that prophage induction is a collateral effect of in-feed antibiotics. IMPORTANCE This study advances our knowledge of the collateral effects of in-feed antibiotics at a time in which the widespread use of “growth-promoting” antibiotics in agriculture is under scrutiny. Using comparative metagenomics, we show that prophages are induced by in-feed antibiotics in swine fecal microbiomes and that antibiotic resistance genes were detected in most viromes. This suggests that in-feed antibiotics are contributing to phage-mediated gene transfer, potentially of antibiotic resistance genes, in the swine gut. Additionally, the so-called “kill-the-winner” model of phage-bacterium population dynamics has been shown in aquatic ecosystems but met with conflicting evidence in gut ecosystems. The data support the idea that swine fecal Streptococcus bacteria and their phages follow the kill-the-winner model. Understanding the role of phages in gut microbial ecology is an essential component of the antibiotic resistance problem and of developing potential mitigation strategies.

Stress Alters Rates and Types of Loss of Heterozygosity in Candida albicans

Abstract: Genetic diversity is often generated during adaptation to stress, and in eukaryotes some of this diversity is thought to arise via recombination and reassortment of alleles during meiosis. Candida albicans, the most prevalent pathogen of humans, has no known meiotic cycle, and yet it is a heterozygous diploid that undergoes mitotic recombination during somatic growth. It has been shown that clinical isolates as well as strains passaged once through a mammalian host undergo increased levels of re- combination. Here, we tested the hypothesis that stress conditions increase rates of mitotic recombination in C. albicans, which is measured as loss of heterozygosity (LOH) at specific loci. We show that LOH rates are elevated during in vitro exposure to oxi- dative stress, heat stress, and antifungal drugs. In addition, an increase in stress severity correlated well with increased LOH rates. LOH events can arise through local recombination, through homozygosis of longer tracts of chromosome arms, or by whole-chromosome homozygosis. Chromosome arm homozygosis was most prevalent in cultures grown under conventional lab conditions. Importantly, exposure to different stress conditions affected the levels of different types of LOH events, with oxida- tive stress causing increased recombination, whilefluconazole and high temperature caused increases in events involving whole chromosomes. Thus, C. albicans generates increased amounts and different types of genetic diversity in response to a range of stress conditions, a process that we term "stress-induced LOH" that arises either by elevating rates of recombination and/or by increasing rates of chromosome missegregation. IMPORTANCE Stress-induced mutagenesis fuels the evolution of bacterial pathogens and is mainly driven by genetic changes via mitotic recombination. Little is known about this process in other organisms. Candida albicans, an opportunistic fungal patho- gen, causes infections that require adaptation to different host environmental niches. We measured the rates of LOH and the types of LOH events that appeared in the absence and in the presence of physiologically relevant stresses and found that stress causes a significant increase in the rates of LOH and that this increase is proportional to the degree of stress. Furthermore, the types of LOH events that arose differed in a stress-dependent manner, indicating that eukaryotic cells generate increased genetic diversity in response to a range of stress conditions. We propose that this "stress-induced LOH" facilitates the rapid adaptation of C. albicans, which does not undergo meiosis, to changing environments within the host.

Microbial Communities of the Upper Respiratory Tract and Otitis Media in Children

Abstract: Streptococcus pneumoniae asymptomatically colonizes the upper respiratory tract of children and is a frequent cause of otitis media. Patterns of microbial colonization likely influence S. pneumoniae colonization and otitis media susceptibility. This study compared microbial communities in children with and without otitis media. Nasal swabs and clinical and demographic data were collected in a cross-sectional study of Philadelphia, PA, children (6 to 78 months) (n = 108) during the 2008-2009 winter respiratory virus season. Swabs were cultured for S. pneumoniae. DNA was extracted from the swabs; 16S rRNA gene hypervariable regions (V1 and V2) were PCR amplified and sequenced by Roche/454 Life Sciences pyrosequencing. Microbial communities were described using the Shannon diversity and evenness indices. Principal component analysis (PCA) was used to group microbial community taxa into four factors representing correlated taxa. Of 108 children, 47 (44%) were colonized by S. pneumoniae, and 25 (23%) were diagnosed with otitis media. Microbial communities with S. pneumoniae were significantly less diverse and less even. Two PCA factors were associated with a decreased risk of pneumococcal colonization and otitis media, as follows: one factor included potentially protective flora (Corynebacterium and Dolosigranulum), and the other factor included Propionibacterium, Lactococcus, and Staphylococcus. The remaining two PCA factors were associated with an increased risk of otitis media. One factor included Haemophilus, and the final factor included Actinomyces, Rothia, Neisseria, and Veillonella. Generally, these taxa are not considered otitis media pathogens but may be important in the causal pathway. Increased understanding of upper respiratory tract microbial communities will contribute to the development of otitis media treatment and prevention strategies. IMPORTANCE Otitis media (middle ear infection) is the most common reason for pediatric sick visits in the United States. Streptococcus pneumoniae is a leading otitis media pathogen. S. pneumoniae must colonize the upper respiratory tract and compete with a complex community of nonpathogenic bacteria before infecting the middle ear. We compared microbial communities in the upper respiratory tract of children who had otitis media and those who did not. Members of the normal flora, i.e., Corynebacterium and Dolosigranulum, were protective for S. pneumoniae colonization and otitis media. As expected, the genera Haemophilus was associated with otitis media. Surprisingly, Actinomyces, Rothia, Neisseria, and Veillonella were associated with an increased risk of otitis media. These bacteria are not otitis media pathogens but may be associated with antibiotic use or involved in the causal pathway to disease. Increased understanding of upper respiratory tract microbial communities will lead to new ways to prevent middle ear infections, including probiotics.

Genome Variation in Cryptococcus gattii, an Emerging Pathogen of Immunocompetent Hosts

Abstract: Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.

Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion

Abstract: A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 10 14 -fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call “enhanced QuIC” (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples. IMPORTANCE Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals.

Molecular Dissection of an Outbreak of Carbapenem-Resistant Enterobacteriaceae Reveals Intergenus KPC Carbapenemase Transmission through a Promiscuous Plasmid

Abstract: Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as major causes of health care-associated infections worldwide. This diverse collection of organisms with various resistance mechanisms is associated with increased lengths of hospitalization, costs of care, morbidity, and mortality. The global spread of CRE has largely been attributed to dissemination of a dominant strain of Klebsiella pneumoniae producing a serine β-lactamase, termed K. pneumoniae carbapenemase (KPC). Here we report an outbreak of KPC-producing CRE infections in which the degree of horizontal transmission between strains and species of a promiscuous plasmid is unprecedented. Sixteen isolates, comprising 11 unique strains, 6 species, and 4 genera of bacteria, were obtained from 14 patients over the first 8 months of the outbreak. Of the 11 unique strains, 9 harbored the same highly promiscuous plasmid carrying the KPC gene bla(KPC). The remaining strains harbored distinct bla(KPC) plasmids, one of which was carried in a strain of Klebsiella oxytoca coisolated from the index patient and the other generated from transposition of the bla(KPC) element Tn4401. All isolates could be genetically traced to the index patient. Molecular epidemiological investigation of the outbreak was aided by the adaptation of nested arbitrary PCR (ARB-PCR) for rapid plasmid identification. This detailed molecular genetic analysis, combined with traditional epidemiological investigation, provides insights into the highly fluid dynamics of drug resistance transmission during the outbreak. IMPORTANCE The ease of horizontal transmission of carbapenemase resistance plasmids across strains, species, and genera of bacteria observed in this study has several important public health and epidemiological implications. First, it has the potential to promote dissemination of carbapenem resistance to new populations of Enterobacteriaceae, including organisms of low virulence, leading to the establishment of reservoirs of carbapenem resistance genes in patients and/or the environment and of high virulence, raising the specter of untreatable community-associated infections. Second, recognition of plasmid-mediated outbreaks, such as those described here, is problematic because analysis of resistance plasmids from clinical isolates is laborious and technically challenging. Adaptation of nested arbitrary PCR (ARB-PCR) to investigate the plasmid outbreak facilitated our investigation, and the method may be broadly applicable to other outbreaks due to other conserved mobile genetic elements. Whether infection control measures that focus on preventing transmission of drug-resistant clones are effective in controlling dissemination of these elements is unknown.

Host- and Strain-Specific Regulation of Influenza Virus Polymerase Activity by Interacting Cellular Proteins

Abstract: Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype have recently emerged from avian zoonotic reservoirs to cause fatal human disease. Adaptation of HPAI virus RNA-dependent RNA polymerase (PB1, PB2, and PA proteins) and nucleoprotein (NP) to interactions with mammalian host proteins is thought to contribute to the efficiency of viral RNA synthesis and to disease severity. While proteomics experiments have identified a number of human proteins that associate with H1N1 polymerases and/or viral ribonucleoprotein (vRNP), how these host interactions might regulate influenza virus polymerase functions and host adaptation has been largely unexplored. We took a functional genomics (RNA interference [RNAi]) approach to assess the roles of a network of human proteins interacting with influenza virus polymerase proteins in viral polymerase activity from prototype H1N1 and H5N1 viruses. A majority (18 of 31) of the cellular proteins tested, including RNA-binding (DDX17, DDX5, NPM1, and hnRNPM), stress (PARP1, DDB1, and Ku70/86), and intracellular transport proteins, were required for efficient activity of both H1N1 and H5N1 polymerases. NXP2 and NF90 antagonized both polymerases, and six more RNA-associated proteins exhibited strain-specific phenotypes. Remarkably, 12 proteins differentially regulated H5N1 polymerase according to PB2 genotype at mammalian-adaptive residue 627. Among these, DEAD box RNA helicase DDX17/p72 facilitated efficient human-adapted (627K) H5N1 virus mRNA and viral RNA (vRNA) synthesis in human cells. Likewise, the chicken DDX17 homologue was required for efficient avian (627E) H5N1 infection in chicken DF-1 fibroblasts, suggesting that this conserved virus-host interaction contributes to PB2-dependent host species specificity of influenza virus and ultimately to the outcome of human HPAI infections. IMPORTANCE Highly pathogenic avian influenza A (HPAI) viruses have recently emerged from wild and domestic birds to cause fatal human disease. In human patients, it is thought that adaptation of the viral polymerase, a complex of viral proteins responsible for viral gene expression and RNA genome replication, to interactions with mammalian rather than avian host proteins contributes to disease severity. In this study, we used computational analysis and RNA interference (RNAi) experiments to identify a biological network of human proteins that regulates an H5N1 HPAI virus polymerase, in comparison to a mammalian H1N1 virus. Of 31 proteins tested, 18 (58%) were required for polymerase function in both HPAI and H1N1 viruses. Remarkably, we also found proteins such as DDX17 that governed the HPAI virus polymerase’s adaptation to human cells. These virus-host interactions may thus control pathogenicity of HPAI virus in humans and are promising therapeutic targets for antiviral drugs in severe influenza infections.

The Sociomicrobiology of Antivirulence Drug Resistance: a Proof of Concept

Abstract: Antivirulence drugs disarm rather than kill pathogens and are thought to alleviate the problem of resistance, although there is no evidence to support this notion. Quorum sensing (QS) often controls cooperative virulence factor production and is therefore an attractive antivirulence target, for which inhibitors (QSI) have been developed. We designed a proof-of-principle experiment to investigate the impact of bacterial social interactions on the evolution of QSI resistance. We cocultured Pseu- domonas aeruginosa QS-deficient mutants with small proportions of the QS-proficient wild type, which in the absence of QSI mimic QSI-sensitive and -resistant variants, respectively. We employed two different QS-dependent nutrients that are degraded by extracellular (public) and cell-associated (private) enzymes. QS mutants (QSI-sensitive mimics) behaved as social cheaters that delayed population growth and prevented enrichment of wild-type cooperators (QSI-resistant mimics) only when nutrient acquisition was public, suggesting that QSI resistance would not spread. This highlights the potential for antivirulence strategies that target cooperative behaviors and provides a conceptual framework for future studies.

Directed Culturing of Microorganisms Using Metatranscriptomics

Abstract: The vast majority of bacterial species remain uncultured, and this severely limits the investigation of their physiology, metabolic capabilities, and role in the environment. High-throughput sequencing of RNA transcripts (RNA-seq) allows the investigation of the diverse physiologies from uncultured microorganisms in their natural habitat. Here, we report the use of RNA-seq for characterizing the metatranscriptome of the simple gut microbiome from the medicinal leech Hirudo verbana and for utilizing this information to design a medium for cultivating members of the microbiome. Expression data suggested that a Rikenella-like bacterium, the most abundant but uncultured symbiont, forages on sulfated- and sialated-mucin glycans that are fermented, leading to the secretion of acetate. Histological stains were consistent with the presence of sulfated and sialated mucins along the crop epithelium. The second dominant symbiont, Aeromonas veronii, grows in two different microenvironments and is predicted to utilize either acetate or carbohydrates. Based on the metatranscriptome, a medium containing mucin was designed, which enabled the cultivation of the Rikenella-like bacterium. Metatranscriptomes shed light on microbial metabolism in situ and provide critical clues for directing the culturing of uncultured microorganisms. By choosing a condition under which the desired organism is rapidly proliferating and focusing on highly expressed genes encoding hydrolytic enzymes, binding proteins, and transporters, one can identify an organism’s nutritional preferences and design a culture medium. IMPORTANCE The number of prokaryotes on the planet has been estimated to exceed 1030 cells, and the overwhelming majority of them have evaded cultivation, making it difficult to investigate their ecological, medical, and industrial relevance. The application of transcriptomics based on high-throughput sequencing of RNA transcripts (RNA-seq) to microorganisms in their natural environment can provide investigators with insight into their physiologies under optimal growth conditions. We utilized RNA-seq to learn more about the uncultured and cultured symbionts that comprise the relatively simple digestive-tract microbiome of the medicinal leech. The expression data revealed highly expressed hydrolytic enzymes and transporters that provided critical clues for the design of a culture medium enabling the isolation of the previously uncultured Rikenella-like symbiont. This directed culturing method will greatly aid efforts aimed at understanding uncultured microorganisms, including beneficial symbionts, pathogens, and ecologically relevant microorganisms, by facilitating genome sequencing, physiological characterization, and genetic manipulation of the previously uncultured microbes.

Streptococcus pneumoniae DNA Initiates Type I Interferon Signaling in the Respiratory Tract

Abstract: The mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogen Streptococcus pneumoniae activates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-β expression through a DAI/STING/TBK1/IRF3 cascade. Tlr4−/−, Myd88−/−, Trif−/−, and Nod2−/− mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-α/β receptor null mice had significantly increased nasal colonization with S. pneumoniae compared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors. IMPORTANCE The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.

The Transformation of Enterovirus Replication Structures: a Three-Dimensional Study of Single- and Double-Membrane Compartments

Abstract: All positive-strand RNA viruses induce membrane structures in their host cells which are thought to serve as suitable microenvironments for viral RNA synthesis. The structures induced by enteroviruses, which are members of the family Picornaviridae , have so far been described as either single- or double-membrane vesicles (DMVs). Aside from the number of delimiting membranes, their exact architecture has also remained elusive due to the limitations of conventional electron microscopy. In this study, we used electron tomography (ET) to solve the three-dimensional (3-D) ultrastructure of these compartments. At different time points postinfection, coxsackievirus B3-infected cells were high-pressure frozen and freeze-substituted for ET analysis. The tomograms showed that during the exponential phase of viral RNA synthesis, closed smooth single-membrane tubules constituted the predominant virus-induced membrane structure, with a minor proportion of DMVs that were either closed or connected to the cytosol in a vase-like configuration. As infection progressed, the DMV number steadily increased, while the tubular single-membrane structures gradually disappeared. Late in infection, complex multilamellar structures, previously unreported, became apparent in the cytoplasm. Serial tomography disclosed that their basic unit is a DMV, which is enwrapped by one or multiple cisternae. ET also revealed striking intermediate structures that strongly support the conversion of single-membrane tubules into double-membrane and multilamellar structures by a process of membrane apposition, enwrapping, and fusion. Collectively, our work unravels the sequential appearance of distinct enterovirus-induced replication structures, elucidates their detailed 3-D architecture, and provides the basis for a model for their transformation during the course of infection. IMPORTANCE Positive-strand RNA viruses hijack specific intracellular membranes and remodel them into special structures that support viral RNA synthesis. The ultrastructural characterization of these “r e plication structures” is key to understanding their precise role. Here, we resolved the three-dimensional architecture of enterovirus-induced membranous compartments and their transformation in time by applying electron tomography to cells infected with coxsackievirus B3 (CVB3). Our results show that closed single-membrane tubules are the predominant initial virus-induced structure, whereas double-membrane vesicles (DMVs) become increasingly abundant at the expense of these tubules as infection progresses. Additionally, more complex multilamellar structures appear late in infection. Based on compelling intermediate structures in our tomograms, we propose a model for transformation from the tubules to DMVs and multilamellar structures via enwrapping events. Our work provides an in-depth analysis of the development of an unsuspected variety of distinct replication structures during the course of CVB3 infection.

Lethal Synergism of 2009 Pandemic H1N1 Influenza Virus and Streptococcus pneumoniae Coinfection Is Associated with Loss of Murine Lung Repair Responses

Abstract: Secondary bacterial infections increase disease severity of influenza virus infections and contribute greatly to increased morbidity and mortality during pandemics. To study secondary bacterial infection following influenza virus infection, mice were inoculated with sublethal doses of 2009 seasonal H1N1 virus (NIH50) or pandemic H1N1 virus (Mex09) followed by inoculation with Streptococcus pneumoniae 48 h later. Disease was characterized by assessment of weight loss and survival, titration of virus and bacteria by quantitative reverse transcription-PCR (qRT-PCR), histopathology, expression microarray, and immunohistochemistry. Mice inoculated with virus alone showed 100% survival for all groups. Mice inoculated with Mex09 plus S. pneumoniae showed severe weight loss and 100% mortality with severe alveolitis, denuded bronchiolar epithelium, and widespread expression of apoptosis marker cleaved caspase 3. In contrast, mice inoculated with NIH50 plus S. pneumoniae showed increased weight loss, 100% survival, and slightly enhanced lung pathology. Mex09-S. pneumoniae coinfection also resulted in increased S. pneumoniae replication in lung and bacteremia late in infection. Global gene expression profiling revealed that Mex09-S. pneumoniae coinfection did not induce significantly more severe inflammatory responses but featured significant loss of epithelial cell reproliferation and repair responses. Histopathological examination for cell proliferation marker MCM7 showed significant staining of airway epithelial cells in all groups except Mex09-S. pneumoniae-infected mice. This study demonstrates that secondary bacterial infection during 2009 H1N1 pandemic virus infection resulted in more severe disease and loss of lung repair responses than did seasonal influenza viral and bacterial coinfection. Moreover, this study provides novel insights into influenza virus and bacterial coinfection by showing correlation of lethal outcome with loss of airway basal epithelial cells and associated lung repair responses. IMPORTANCE Secondary bacterial pneumonias lead to increased disease severity and have resulted in a significant percentage of deaths during influenza pandemics. To understand the biological basis for the interaction of bacterial and viral infections, mice were infected with sublethal doses of 2009 seasonal H1N1 and pandemic H1N1 viruses followed by infection with Streptococcus pneumoniae 48 h later. Only infection with 2009 pandemic H1N1 virus and S. pneumoniae resulted in severe disease with a 100% fatality rate. Analysis of the host response to infection during lethal coinfection showed a significant loss of responses associated with lung repair that was not observed in any of the other experimental groups. This group of mice also showed enhanced bacterial replication in the lung. This study reveals that the extent of lung damage during viral infection influences the severity of secondary bacterial infections and may help explain some differences in mortality during influenza pandemics.

From Commensal to Pathogen: Translocation of Enterococcus faecalis from the Midgut to the Hemocoel of Manduca sexta

Abstract: A dynamic homeostasis is maintained between the host and native bacteria of the gastrointestinal tract in animals, but migration of bacteria from the gut to other organs can lead to disease or death. Enterococcus faecalis is a commensal of the gastrointestinal tract; however, Enterococcus spp. are increasingly frequent causes of nosocomial infections with a high mortality rate. We investigated the commensal-to-pathogen switch undergone by E. faecalis OG1RF in the lepidopteran model host Manduca sexta associated with its location in the host. E. faecalis persists in the harsh midgut environment of M. sexta larvae without causing apparent illness, but injection of E. faecalis directly into the larval hemocoel is followed by rapid death. Additionally, oral ingestion of E. faecalis in the presence of Bacillus thuringiensis insecticidal toxin, a pore-forming toxin that targets the midgut epithelium, induces an elevated mortality rate. We show that the loss of gut integrity due to B. thuringiensis toxin correlates with the translocation of E. faecalis from the gastrointestinal tract into the hemolymph. Upon gaining access to the hemolymph, E. faecalis induces an innate immune response, illustrated by hemocyte aggregation, in larvae prior to death. The degree of hemocyte aggregation is dependent upon the route of E. faecalis entry. Our data demonstrate the efficacy of the M. sexta larval model system in investigating E. faecalis-induced sepsis and clarifies controversies in the field regarding the events leading to larval death following B. thuringiensis toxin exposure. IMPORTANCE This study advances our knowledge of Enterococcus faecalis-induced sepsis following translocation from the gut and provides a model for mammalian diseases in which the spatial distribution of bacteria determines disease outcomes. We demonstrate that E. faecalis is a commensal in the gut of Manduca sexta and a pathogen in the hemocoel, resulting in a robust immune response and rapid death, a process we refer to as the “commensal-to-pathogen” switch. While controversy remains regarding Bacillus thuringiensis toxin-induced killing, our laboratory previously found that under some conditions, the midgut microbiota is essential for B. thuringiensis toxin killing of Lymantria dispar (N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196–15199, 2006; B. Raymond, et al., Environ. Microbiol. 11:2556–2563, 2009; P. R. Johnston, and N. Crickmore, Appl. Environ. Microbiol. 75:5094–5099, 2009). We and others have demonstrated that the role of the midgut microbiota in B. thuringiensis toxin killing is dependent upon the lepidopteran species and formulation of B. thuringiensis toxin (N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196–15199, 2006; N. A. Broderick, et al., BMC Biol. 7:11, 2009). This work reconciles much of the apparently contradictory previous data and reveals that the M. sexta-E. faecalis system provides a model for mammalian sepsis.

Necrotic Enteritis-Derived Clostridium perfringens Strain with Three Closely Related Independently Conjugative Toxin and Antibiotic Resistance Plasmids

Abstract: The pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates of Clostridium perfringens type A. To determine the location and mobility of the netB structural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in which netBwas insertionally inactivated by the chloramphen- icol and thiamphenicol resistance gene catP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and the netBgene were present on different conjugative ele- ments. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the re- sultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 from C. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conju- gative 82-kb plasmid that contained the netBgene and other potential virulence genes, and (iii) a 70-kb plasmid that carried the cpb2gene, which encodes a different pore-forming toxin, beta2 toxin. IMPORTANCE The anaerobic bacterium Clostridium perfringens can cause an avian gastrointestinal disease known as necrotic enteritis. Disease pathogenesis is not well understood, although the plasmid-encoded pore-forming toxin NetB, is an important virulence factor. In this work, we have shown that the plasmid that carries the netBgene is conjugative and has a 40-kb region that is very similar to replication and transfer regions found within each of the sequenced conjugative plasmids from C. perfrin- gens. We also showed that this strain contained two additional large plasmids that were also conjugative and carried a similar 40-kb region. One of these plasmids encoded beta2 toxin, and the other encoded tetracycline resistance. To our knowledge, this is thefirst report of a bacterial strain that carries three closely related but different independently conjugative plasmids. These results have significant implications for our understanding of the transmission of virulence and antibiotic resistance genes in pathogenic bacteria.

ChePep Controls Helicobacter pylori Infection of the Gastric Glands and Chemotaxis in the Epsilonproteobacteria

Abstract: Microbes use directed motility to colonize harsh and dynamic environments. We discovered that Helicobacter pylori strains establish bacterial colonies deep in the gastric glands and identified a novel protein, ChePep, necessary to colonize this niche. ChePep is preferentially localized to the flagellar pole. Although mutants lacking ChePep have normal flagellar ultrastructure and are motile, they have a slight defect in swarming ability. By tracking the movement of single bacteria, we found that ∆ChePep mutants cannot control the rotation of their flagella and swim with abnormally frequent reversals. These mutants even sustain bursts of movement backwards with the flagella pulling the bacteria. Genetic analysis of the chemotaxis signaling pathway shows that ChePep regulates flagellar rotation through the chemotaxis system. By examining H. pylori within a microscopic pH gradient, we determined that ChePep is critical for regulating chemotactic behavior. The chePep gene is unique to the Epsilonproteobacteria but is found throughout this diverse group. We expressed ChePep from other members of the Epsilonproteobacteria , including the zoonotic pathogen Campylobacter jejuni and the deep sea hydrothermal vent inhabitant Caminibacter mediatlanticus, in H. pylori and found that ChePep is functionally conserved across this class. ChePep represents a new family of chemotaxis regulators unique to the Epsilonproteobacteria and illustrates the different strategies that microbes have evolved to control motility. IMPORTANCE Helicobacter pylori strains infect half of all humans worldwide and contribute to the development of peptic ulcers and gastric cancer. H. pylori cannot survive within the acidic lumen of the stomach and uses flagella to actively swim to and colonize the protective mucus and epithelium. The chemotaxis system allows H. pylori to navigate by regulating the rotation of its flagella. We identified a new protein, ChePep, which controls chemotaxis in H. pylori. ChePep mutants fail to colonize the gastric glands of mice and are completely outcompeted by normal H. pylori. Genes encoding ChePep are found only in the class Epsilonproteobacteria , which includes the human pathogen Campylobacter jejuni and environmental microbes like the deep-sea hydrothermal vent colonizer Caminibacter mediatlanticus, and we show that ChePep function is conserved in this class. Our study identifies a new colonization factor in H. pylori and also provides insight into the control and evolution of bacterial chemotaxis.

Nonlytic Exocytosis of Cryptococcus neoformans from Macrophages Occurs In Vivo and Is Influenced by Phagosomal pH

Abstract: A unique aspect of the interaction of the fungus Cryptococcus neoformans with macrophages is the phenomenon of nonlytic exocytosis, also referred to as "vomocytosis" or phagosome extrusion/expulsion, which involves the escape of fungal cells from the phagocyte with the survival of both cell types. This phenomenon has been observed only in vitro using subjective and time-consuming microscopic techniques. In spite of recent advances in our knowledge about its mechanisms, a major ques- tion still remaining is whether this phenomenon also occurs in vivo. In this study, we describe a novel flow cytometric method that resulted in a substantial gain in throughput for studying phagocytosis and nonlytic exocytosis in vitro and used it to explore the occurrence of this phenomenon in a mouse model of infection. Furthermore, we tested the hypothesis that host cell phago- somal pH affected nonlytic exocytosis. The addition of the weak bases ammonium chloride and chloroquine resulted in a signifi- cant increase of nonlytic exocytosis events, whereas the vacuolar ATPase inhibitor bafilomycin A1 had the opposite effect. Al- though all three agents are known to neutralize phagosomal acidity, their disparate effects suggest that phagosomal pH is an important and complex variable in this process. Our experiments established that nonlytic exocytosis occurred in vivo with a frequency that is possibly much higher than that observed in vitro. These results in turn suggest that nonlytic exocytosis has a potential role in the pathogenesis of cryptococcosis. IMPORTANCE Cryptococcus neoformans causes disease in people with immune deficiencies such as AIDS. Upon infection, C. neo- formans cells are ingested by macrophage immune cells, which provide a niche for survival and replication. After ingestion, mac- rophages can expel the fungi without causing harm to either cell type, a process named nonlytic exocytosis. To dissect this phe- nomenon, we evaluated its dependence on the pH inside the macrophage and addressed its occurrence during infection of mice. We developed new techniques using flow cytometry to measure C. neoformans internalization by and nonlytic exocytosis from macrophages. Neutralizing the phagosome acidity changed the rate of nonlytic exocytosis: activity increased with the weak bases chloroquine and ammonium chloride, whereas the vacuolar ATPase inhibitor bafilomycin A1 caused it to decrease. Experiments in mice suggested that nonlytic exocytosis occurred during infection with C. neoformans. These results shed new light on the interaction between C. neoformans and host macrophages.

Cellular Architecture Mediates DivIVA Ultrastructure and Regulates Min Activity in Bacillus subtilis

Abstract: The assembly of the cell division machinery at midcell is a critical step of cytokinesis. Many rod-shaped bacteria position septa using nucleoid occlusion, which prevents division over the chromosome, and the Min system, which prevents division near the poles. Here we examined the in vivo assembly of the Bacillus subtilis MinCD targeting proteins DivIVA, a peripheral membrane protein that preferentially localizes to negatively curved membranes and resembles eukaryotic tropomyosins, and MinJ, which recruits MinCD to DivIVA. We used structured illumination microscopy to demonstrate that both DivIVA and MinJ localize as double rings that flank the septum and first appear early in septal biosynthesis. The subsequent recruitment of MinCD to these double rings would separate the Min proteins from their target, FtsZ, spatially regulating Min activity and allowing continued cell division. Curvature-based localization would also provide temporal regulation, since DivIVA and the Min proteins would localize to midcell after the onset of division. We use time-lapse microscopy and fluorescence recovery after photobleaching to demonstrate that DivIVA rings are highly stable and are constructed from newly synthesized DivIVA molecules. After cell division, DivIVA rings appear to collapse into patches at the rounded cell poles of separated cells, with little or no incorporation of newly synthesized subunits. Thus, changes in cell architecture mediate both the initial recruitment of DivIVA to sites of cell division and the subsequent collapse of these rings into patches (or rings of smaller diameter), while curvature-based localization of DivIVA spatially and temporally regulates Min activity. IMPORTANCE The Min systems of Escherichia coli and Bacillus subtilis both inhibit FtsZ assembly, but one key difference between these two species is that whereas the E. coli Min proteins localize to the poles, the B. subtilis proteins localize to nascent division sites by interaction with DivIVA and MinJ. It is unclear how MinC activity at midcell is regulated to prevent it from interfering with FtsZ engaged in medial cell division. We used superresolution microscopy to demonstrate that DivIVA and MinJ, which localize MinCD, assemble double rings that flank active division sites and septa. This curvature-based localization mechanism holds MinCD away from the FtsZ ring at midcell, and we propose that this spatial organization is the primary mechanism by which MinC activity is regulated to allow division at midcell. Curvature-based localization also conveys temporal regulation, since it ensures that MinC localizes after the onset of division.

A Systems Biology Approach to Infectious Disease Research: Innovating the Pathogen-Host Research Paradigm

Abstract: The twentieth century was marked by extraordinary advances in our understanding of microbes and infectious disease, but pandemics remain, food and waterborne illnesses are frequent, multidrug-resistant microbes are on the rise, and the needed drugs and vaccines have not been developed. The scientific approaches of the past-including the intense focus on individual genes and proteins typical of molecular biology-have not been sufficient to address these challenges. The first decade of the twenty-first century has seen remarkable innovations in technology and computational methods. These new tools provide nearly comprehensive views of complex biological systems and can provide a correspondingly deeper understanding of pathogen-host interactions. To take full advantage of these innovations, the National Institute of Allergy and Infectious Diseases recently initiated the Systems Biology Program for Infectious Disease Research. As participants of the Systems Biology Program, we think that the time is at hand to redefine the pathogen-host research paradigm.

Evidence of a Dominant Lineage of Vibrio cholerae-Specific Lytic Bacteriophages Shed by Cholera Patients over a 10-Year Period in Dhaka, Bangladesh

Abstract: Lytic bacteriophages are hypothesized to contribute to the seasonality and duration of cholera epidemics in Bangladesh. However, the bacteriophages contributing to this phenomenon have yet to be characterized at a molecular genetic level. In this study, we isolated and sequenced the genomes of 15 bacteriophages from stool samples from cholera patients spanning a 10-year surveillance period in Dhaka, Bangladesh. Our results indicate that a single novel bacteriophage type, designated ICP1 (for the International Centre for Diarrhoeal Disease Research, Bangladesh cholera phage 1) is present in all stool samples from cholera patients, while two other bacteriophage types, one novel (ICP2) and one T7-like (ICP3), are transient. ICP1 is a member of the Myoviridae family and has a 126-kilobase genome comprising 230 open reading frames. Comparative sequence analysis of ICP1 and related isolates from this time period indicates a high level of genetic conservation. The ubiquitous presence of ICP1 in cholera patients and the finding that the O1 antigen of lipopolysaccharide (LPS) serves as the ICP1 receptor suggest that ICP1 is extremely well adapted to predation of human-pathogenic V. cholerae O1. IMPORTANCE The severe diarrheal disease cholera is caused by the bacterium Vibrio cholerae, which can be transmitted to humans from the aquatic environment. Factors that affect V. cholerae in the environment can impact the occurrence of cholera outbreaks; one of these factors is thought to be the presence of bacterial viruses, or bacteriophages. Bacteriophages that prey on V. cholerae in the environment, and potentially in humans, have not been extensively genetically characterized. Here, we isolated and sequenced the genomes of bacteriophages from cholera patient stool samples collected over a 10-year period in Dhaka, Bangladesh, a region that suffers from regular cholera outbreaks. We describe a unique bacteriophage present in all samples, infer its evolution by sequencing multiple isolates from different patients over time, and identify the host receptor that shows that the bacteriophage specifically predates the serogroup of V. cholerae responsible for the majority of disease occurrences.