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bioRxiv2017

Sofosbuvir protects Zika virus-infected mice from mortality, preventing short- and long-term sequela

Zika virus (ZIKV) caused significant public health concern, because of its association with congenital malformations, neurological disorders in adults and, more recently, with deaths. Considering the necessity to mitigate the cases ZIKV-associated diseases, antiviral interventions against this virus are an urgent necessity. Sofosbuvir, a drug in clinical use against Hepatitis C Virus (HCV), is among the FDA-approved substances endowed with anti-ZIKV activity. In this work, we further investigated the in vivo activity of sofosbuvir against ZIKV. Neonatal Swiss mice were infected with ZIKV (2 x 107 PFU) and treated with sofosbuvir at 20 mg/kg/day, a concentration compatible with pre-clinical development of this drug. We found that sofosbuvir reduced acute levels of ZIKV from 60 to 90 % in different anatomical compartments, such as in blood plasma, spleen, kidney and brain. Early treatment with sofosbuvir doubled the percentage and time of survival of ZIKV-infected animals, despite the aggressive virus challenge assayed and also prevented the acute neuromotor impairment triggered by the virus. On the long-term behavior analysis of ZIKV-associated sequelae, sofosbuvir prevented loss of hippocampal- and amygdala-dependent memory. Our results point out that sofosbuvir inhibits ZIKV replication in vivo, which is consistent with the prospective necessity of antiviral drugs to treat ZIKV-infected individuals.

microbiology↗

bioRxiv2017

Pooled CRISPR interference screens enable high-throughput functional genomics study and elucidate new rules for guide RNA library design in Escherichia coli

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology provides potential advantages in high-throughput functional genomics analysis in prokaryotes over previously established platforms based on recombineering or transposon mutagenesis. In this work, as a proof-of-concept to adopt CRISPR/Cas9 method as a pooled functional genomics analysis platform in prokaryotes, we developed a CRISPR interference (CRISPRi) library consisting of 3,148 single guide RNAs (sgRNAs) targeting the open reading frame (ORF) of 67 genes with known knockout phenotypes and performed pooled screens under two stressed conditions (minimal and acidic medium) in Escherichia coli. Our approach confirmed most of previously described gene-phenotype associations while maintaining < 5% false positive rate, suggesting that CRISPRi screen is both sensitive and specific. Our data also supported the ability of this method to narrow down the candidate gene pool when studying operons, a unique structure in prokaryotic genome. Meanwhile, assessment of multiple loci across treatments enables us to extract several guidelines for sgRNA design for such pooled functional genomics screen. For instance, sgRNAs locating at the first 5% upstream region within ORF exhibit enhanced activity and 10 sgRNAs per gene is suggested to be enough for robust identification of gene-phenotype associations. We also optimized the hit-gene calling algorithm to identify target genes more robustly with even fewer sgRNAs. This work showed that CRISPRi could be adopted as a powerful functional genomics analysis tool in prokaryotes and provided the first guideline for the construction of sgRNA libraries in such applications.\n\nImportanceTo fully exploit the valuable resource of explosive sequenced microbial genomes, high-throughput experimental platform is needed to associate genes and phenotypes at the genome level, giving microbiologists the insight about the genetic structure and physiology of a microorganism. In this work, we adopted CRISPR interference method as a pooled high-throughput functional genomics platform in prokaryotes with Escherichia coli as the model organism. Our data suggested that this method was highly sensitive and specific to map genes with previously known phenotypes, potent to act as a new strategy for high-throughput microbial genetics study with advantages over previously established methods. We also provided the first guideline for the sgRNA library design by comprehensive analysis of the screen data. The concept, gRNA library design rules and open-source scripts of this work should benefit prokaryotic genetics community to apply high-throughput mapping of defined gene set with phenotypes in a broad spectrum of microorganisms.

microbiology↗

bioRxiv2017

Nitrogen-Fixing Populations Of Planctomycetes And Proteobacteria Are Abundant In The Surface Ocean

Nitrogen fixation in the surface ocean impacts the global climate by regulating the microbial primary productivity and the sequestration of carbon through the biological pump. Cyanobacterial populations have long been thought to represent the main suppliers of the bio-available nitrogen in this habitat. However, recent molecular surveys of nitrogenase reductase gene revealed the existence of rare non-cyanobacterial populations that can also fix nitrogen. Here, we characterize for the first time the genomic content of some of these heterotrophic bacterial diazotrophs (HBDs) inhabiting the open surface ocean waters. They represent new lineages within Planctomycetes and Proteobacteria, a phylum never linked to nitrogen fixation prior to this study. HBDs were surprisingly abundant in the Pacific Ocean and the Atlantic Ocean northwest, conflicting with decades of PCR surveys. The abundance and widespread occurrence of non-cyanobacterial diazotrophs in the surface ocean emphasizes the need to re-evaluate their role in the nitrogen cycle and primary productivity.

microbiology↗

bioRxiv2017

Interactions Between Molecular Chaperone P20 And Cyt2Ba7 Toxin In Bacillus thuringiensis

P20 or 20-kilodalton protein is a molecular chaperone protein in Bacillus thuringiensis (Bt) which can increase yields and facilitates crystal formation of various insecticidal crystal proteins (ICPs). In previous studies, a B. thuringiensis insecticidal protein gene, cyt2Ba7, was cloned, expressed but its expression level is very low in B. thuringiensis. In this study, various expression vectors were constructed by incorporating p20 in forward or reverse direction in the upstream of cyt2Ba7 and transformed into a B. thuringiensis acrystalliferous strain 4Q7. The result showed that in the presence of P20, the expression of Cyt2Ba7 was significantly increased. Especially when p20 gene was reversely inserted in the upstream of cyt2Ba7 gene, the expression of Cyt2Ba7 was increased [~]3.2 times meanwhile more and bigger crystals were observed under electron microscopy. By using purified Cyt2Ba7, P20 protein and P20-specific antiserum, immunoblotting and ligand blot analysis demonstrated a strong binding affinity between P20 and Cyt2Ba7. These results reveal that P20 can promote the crystal formation and enhance the expression of Cyt2Ba7 as a molecular chaperone, which can be a powerful tool to boost the ICPs production in B. thuringiensis and help develop more effective insect control strategies.

microbiology↗

bioRxiv2017

Extracellular Electron Transfer Powers Enterococcus faecalis Biofilm Metabolism

Enterococci are important human commensals and significant opportunistic pathogens associated with endocarditis, urinary tract infections, wound and surgical site infections, and medical device associated infections. These infections often become chronic upon the formation of biofilm. The biofilm matrix establishes properties that distinguish this state from free-living bacterial cells and increase tolerance to antimicrobial interventions. The metabolic versatility of the Enterococci is reflected in the diversity and complexity of environments and communities in which they thrive. Understanding metabolic factors governing colonization and persistence in different host niches can reveal factors influencing the transition from commensal to opportunistic pathogen. Here, we report a new form of iron-dependent metabolism for Enterococcus faecalis where, in the absence of heme, respiration components can be utilised for extracellular electron transfer (EET). Iron augments E. faecalis biofilm growth and generates alterations in biofilm matrix, cell spatial distribution, and biofilm matrix properties. We identify the genes involved in iron-augmented biofilm growth and show that it occurs by promoting EET to iron within biofilm.\n\nSignificanceBacterial metabolic versatility is often key in dictating the outcome of host-pathogen interactions, yet determinants of metabolic shifts are difficult to resolve. The bacterial biofilm matrix provides the structural and functional support that distinguishes this state from free-living bacterial cells. Here, we show that the biofilm matrix provides access to resources necessary for metabolism and growth which are otherwise inaccessible in the planktonic state. Our data shows that in the absence of heme, components of Enterococcus faecalis respiration (l-lactate dehydrogenase and acetaldehyde dehydrogenase) may function as initiators of EET through the cytoplasmic membrane quinone pool and utilize matrix-associated iron to carry out EET. The presence of iron resources within the biofilm matrix leads to enhanced biofilm growth.

microbiology↗

bioRxiv2017

Modulation Of Type IV Pili Phenotypic Plasticity Through A Novel Chaperone-Usher System In Synechocystis sp 6803

Controlling the transition from a multicellular motile state to a sessile biofilm is an important eco-physiological decision for most prokaryotes, including cyanobacteria. Photosynthetic and bio geochemically significant cyanobacterium Synechocystis sp. PCC6803 (Syn6803) uses Type IV pili (TFP) for surface-associated motility and light-directed phototaxis. We report the identification of a novel Chaperone-Usher (CU) system in Syn6803 that regulate secretion of minor pilins as a means of stabilizing TFP morphology. These secreted minor-pilins aid in modifying TFP morphology to suit the adhesion state by forming cell to surface contacts when motility is not required. This morphotype is structurally distinct from TFP assembled during motile phase. We further demonstrate by examining mutants lacking either the CU system or the minor-pilins, which produce aberrant TFP, that are morphologically and functionally distinct from wild-type (WT). Thus, here we report that in Syn6803, CU system work independent of TFP biogenesis machinery unlike reported for other pathogenic bacterial systems and contributes to provide multifunctional plasticity to TFP. cAMP levels play an important role in controlling this switch. This phenotypic plasticity exhibited by the TFP, in response to cAMP levels would allow cells and cellular communities to adapt to rapidly fluctuating environments by dynamically transitioning between motile and sessile states.\n\nSignificance of this workHow cyanobacterial communities cope with fluctuating or extreme environments is crucial in understanding their role in global carbon and nitrogen cycles. This work addresses the key question: how do cyanobacteria modulate external appendages, called Type IV pili, to effectively switch between motile and sessile biofilm states? We demonstrate that cells transition between forming strong cell-surface interactions indispensable for biofilm formation to forming cell-cell interactions that allow for coordinated movement crucial for social motility by functional/ structural modification of same TFP appendage. The second messenger, cAMP and a Chaperone-Usher secretion are indispensible to achieve these structural modifications of TFP and control the complex phenotypic transition. We have uncovered a strategy that Syn6803 has evolved to deal with molecular decision-making under uncertainty, which we call phenotypic plasticity. Here we demonstrate how a single motility appendage can be structurally modified to attain two antagonistic functions in order to meet the fluctuating environmental demands.

microbiology↗

bioRxiv2017

Assessing species biomass contributions in microbial communities via metaproteomics

Assessment of microbial community composition is the cornerstone of microbial ecology. Microbial community composition can be analyzed by quantifying cell numbers or by quantifying biomass for individual populations. However, as cell volumes can differ by orders of magnitude, these two approaches yield vastly different results. Methods for quantifying cell numbers are already available (e.g. fluorescence in situ hybridization, 16S rRNA gene amplicon sequencing), yet methods for assessing community composition in terms of biomass are lacking.\n\nWe developed metaproteomics based methods for assessing microbial community composition using protein abundance as a measure for biomass contributions of individual populations. We optimized the accuracy and sensitivity of the method using artificially assembled microbial communities and found that it is less prone to some of the biases found in sequencing-based methods. We applied the method using communities from two different environments, microbial mats from two alkaline soda lakes and saliva from multiple individuals.

microbiology↗

bioRxiv2017

Bacterial Biofilm Formation On Soil Fungi: A Widespread Ability Under Controls

In natural environments, bacteria preferentially live in biofilms that they build on abiotic surfaces but also on living tissues. Although fungi form extensive networks of hyphae within soils and thus could provide immense surfaces for bacteria to build biofilms and to proliferate, the extent on such phenomenon and the consequences for the fitness of both microorganisms is poorly known in soils. Here, we analyzed the process of formation of biofilms by various bacteria on hyphae of soil fungi in an in vitro setting using confocal and electron microscopy. We showed that the ability to form biofilms on fungal hyphae is widely shared among soil bacteria. In contrast, some fungi, mainly belonging to the Ascomycete class, did not allow for the formation of bacterial biofilms on their surfaces. The formation of biofilms was also strongly modulated by the presence of tree roots and by the development of the ectomycorrhizal symbiosis, suggesting that biofilm formation does not occur randomly in soil but that it is highly regulated by several biotic factors. Finally, our study led to the unexpected finding that networks of filaments made of extracellular DNA were used to build the skeleton of biofilms by a large array of bacteria.

microbiology↗

bioRxiv2017

High Activity Of Selective Essential Oils Against Stationary Phase Borrelia burgdorferi As A Persister Model

Although the majority of patients with Lyme disease can be cured with the standard 2-4 week antibiotic treatment, about 10-20% of patients continue to suffer from post-treatment Lyme disease syndrome (PTLDS). While the cause for this is debated, one possibility is due to persisters not killed by the current Lyme antibiotics. It has been reported that essential oils have antimicrobial activities and some have been used by patients with persisting Lyme disease symptoms. However, the activity of essential oils against the causative agent Borrelia burgdorferi (B. burgdorferi) has not been carefully studied. Here, we evaluated the activity of 34 essential oils against B. burgdorferi stationary phase culture as a model for persisters. We found that many essential oils had varying degrees of activity against B. burgdorferi, with top 5 essential oils (oregano, cinnamon bark, clove bud, citronella, and wintergreen) at a low concentration of 0.25% showing more activity than the persister drug daptomycin. Interestingly, some highly active essential oils were found to have excellent anti-biofilm ability as shown by their ability to dissolve the aggregated biofilm-like structures. The top 3 hits, oregano, cinnamon bark and clove bud, completely eradicated all viable cells without regrowth in subculture. Carvacrol was found to be the most active ingredient of oregano oil showing excellent activity against B. burgdorferi stationary phase cells, while p-cymene and -terpinene had no apparent activity. Future studies are needed to characterize and optimize the active essential oils in drug combinations in vitro and in vivo for improved treatment of persistent Lyme disease.\n\nIMPORTANCEThere is a huge need for effective treatment of patients with Lyme disease who suffer from PTLDS. Recent in vitro and in vivo studies suggest that B. burgdorferi develops persisters that are not killed by the current Lyme antibiotics as a possible contributor to this condition. Although essential oils are used by patients with Lyme disease with variable improvement in symptoms, their anti-borrelia activity has not been carefully studied. Here we found that not all essential oils have adequate anti-borrelia activity and identified some highly potent essential oils (oregano, cinnamon bark, clove bud) that have even higher anti-persister and anti-biofilm activity than the persister drug daptomycin. Carvacrol was found to be the most active ingredient of oregano oil and have the potential to serve as a promising oral persister drug. Our findings may have implications for developing improved treatment of persisting Lyme disease.

microbiology↗

bioRxiv2017

Dengue virus hijacks a noncanonical oxidoreductase function of a cellular oligosaccharyltransferase complex

Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide CRISPR screen to identify host dependency factors required for DENV propagation, and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CxxC active site motif of MAGT1 was necessary for DENV propagation as cells expressing an AxxA MAGT1 mutant were unable to support DENV infection.\n\nInterestingly, cells expressing single-cysteine CxxA or AxxC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antivirals targeting DENV.

microbiology↗

bioRxiv2017

The human oral microbiome is shaped by shared environment rather than genetics: evidence from a large family of closely-related individuals

The human microbiome is affected by multiple factors, including the environment and host genetics. In this study, we analyzed the oral microbiome of an extended family of Ashkenazi Jewish individuals living in several cities and investigated associations with both shared household and host genetic similarities. We found that environmental effects dominated over genetic ones. While there was weak evidence of geographic structuring at the level of cities, we observed a large and significant effect of shared household on microbiome composition, supporting the role of immediate shared environment in dictating the presence or absence of taxa. This effect was also seen when including adults who had grown up in the same household but moved out prior to the time of sampling, suggesting that the establishment of the oral microbiome earlier in life may affect its long-term composition. We found weak associations between host genetic relatedness and microbiome dissimilarity when using family pedigrees as proxies for genetic similarity. However this association disappeared when using more accurate measures of kinship based on genome-wide genetic markers, indicating that environment rather than host genetics is the dominant factor affecting the composition of the oral microbiome in closely-related individuals. Our results support the concept that there is a consistent core microbiome conserved across global scales, but that small-scale effects due to shared living environment significantly affect microbial community composition.\n\nIMPORTANCEPrevious research shows that relatives have a more similar oral microbiome composition than non-relatives, but it remains difficult to distinguish the effects of relatedness and shared household environment. Furthermore, pedigree measures may not accurately measure host genetic similarity. In this study, we include genetic relatedness based on genome-wide SNPs (rather than pedigree measures) and shared environment in the same analysis. We quantify the relative importance of these factors by studying the oral microbiome in members of a large extended Ashkenazi Jewish family who share a similar diet and lifestyle despite living in different locations. We find that host genetics plays no significant role and that the dominant factor is shared environment at the household level. We also find that this effect appears to persist in individuals who have moved out of the parental household, suggesting that the oral microbiome established earlier in life persists long-term.

microbiology↗

bioRxiv2017

Relic DNA contributes minimally to estimates of microbial diversity

Extracellular or \"relic\" DNA is one of the largest pools of nucleic acids in the mbiosphere 1,2. Relic DNA can influence a number of important ecological and evolutionary processes, but it may also bias estimates of microbial abundance and diversity, which has implications for understanding environmental, engineered, and host-associated ecosystems. We developed models capturing the fundamental processes that regulate the size and composition of the relic DNA pools to identify scenarios leading to biased estimates of biodiversity. Our models predict that bias increases with relic DNA pool size, but only when the species abundance distributions (SAD) of relic and intact DNA are distinct from one another. We evaluated our model predictions by quantifying relic DNA and assessing its contribution to bacterial diversity using 16S rRNA gene sequences collected from different ecosystem types, including soil, sediment, water, and the mammalian gut. On average, relic DNA made up 33 % of the total bacterial DNA pool, but exceeded 80 % in some samples. Despite its abundance, relic DNA had no effect on estimates of taxonomic and phylogenetic diversity, even in ecosystems where processes such as the physical protection of relic DNA are common and predicted by our models to generate bias. Rather, our findings are consistent with the expectation that relic DNA sequences degrade in proportion to their abundance and therefore may contribute minimally to estimates of microbial diversity.

microbiology↗

bioRxiv2017

Epistatic interactions within the influenza virus polymerase complex mediate mutagen resistance and replication fidelity

Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza virus using three nucleoside analogs as well as the viruss high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C to U and G to A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza polymerase, we find that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin-resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates.\n\nImportanceRNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis.

microbiology↗

bioRxiv2017

Assessment Of The Potential Role Of Streptomyces In Cave Moonmilk Formation

Moonmilk is a karstic speleothem mainly composed of fine calcium carbonate crystals (CaCO3) with different textures ranging from pasty to hard, in which the contribution of biotic rock-building processes is presumed to involve indigenous microorganisms. The real bacterial input in the genesis of moonmilk is difficult to assess leading to controversial hypotheses explaining the origins and the mechanisms (biotic versus abiotic) involved. In this work we undertook a comprehensive approach in order to assess the potential role of filamentous bacteria, particularly a collection of moonmilk-originating Streptomyces, in the genesis of this speleothem. Scanning electron microscopy (SEM) confirmed that indigenous filamentous bacteria could indeed participate in moonmilk development by serving as nucleation sites for CaCO3 deposition. The metabolic activities involved in CaCO3 transformation were furthermore assessed in vitro among the collection of moonmilk Streptomyces, which revealed that peptides/amino acids ammonification, and to a lesser extend ureolysis, could be privileged metabolic pathways participating in carbonate precipitation by increasing the pH of the bacterial environment. Additionally, in silico search for the genes involved in biomineralization processes including ureolysis, dissimilatory nitrate reduction to ammonia, active calcium ion transport, and reversible hydration of CO2 allowed to identify genetic predispositions for carbonate precipitation in Streptomyces. Finally, their biomineralization abilities were confirmed by environmental SEM, which allowed to visualize the formation of abundant mineral deposits under laboratory conditions. Overall, our study provides novel evidences that filamentous Actinobacteria could be key protagonists in the genesis of moonmilk through a wide spectrum of biomineralization processes.

microbiology↗

bioRxiv2017

The effector TepP mediates the recruitment and activation of Phosphoinositide 3 Kinase on early Chlamydia trachomatis vacuoles

Chlamydia trachomatis delivers multiple Type 3 secreted effector proteins to host epithelial cells to manipulate cytoskeletal functions, membrane dynamics and signaling pathways. TepP is the most abundant effector protein secreted early in infection but its molecular function is poorly understood. In this report, we provide evidence that TepP is important for bacterial replication in cervical epithelial cells, the activation of Type I IFN genes, and the recruitment of Class I phosphoinositide 3 kinases (PI3K) and the signaling adaptor protein CrkL to nascent pathogen-containing vacuoles (inclusions). We also show that TepP is a target of tyrosine phosphorylation by Src kinases but these modifications do not appear to influence the recruitment of PI3K or CrkL. The translocation of TepP correlated with an increase in the intracellular pools of phosphoinositide 3,4,5 triphosphate but not the activation of the pro-survival kinase Akt, suggesting that TepP-mediated activation of PI3K is spatially restricted to early inclusions. Furthermore, we linked PI3K activity to the dampening of transcription of Type I IFN induced genes early in infection. Overall, these findings indicate that TepP can modulate cell signaling and potentially membrane trafficking events by spatially restricted activation of PI3K.

microbiology↗

bioRxiv2017

A Supervised Statistical Learning Approach For Accurate Legionella pneumophila Source Attribution During Outbreaks

Public health agencies are increasingly relying on genomics during Legionnaires disease investigations. However, the causative bacterium (Legionella pneumophila) has an unusual population structure with extreme temporal and spatial genome sequence conservation. Furthermore, Legionnaires disease outbreaks can be caused by multiple L. pneumophila genotypes in a single source. These factors can confound cluster identification using standard phylogenomic methods. Here, we show that a statistical learning approach based on\n\nL. pneumophila core genome single nucleotide polymorphism (SNP) comparisons eliminates ambiguity for defining outbreak clusters and accurately predicts exposure sources for clinical cases. We illustrate the performance of our method by genome comparisons of 234 L. pneumophila isolates obtained from patients and cooling towers in Melbourne, Australia between 1994 and 2014. This collection included one of the largest reported Legionnaires disease outbreaks, involving 125 cases at an aquarium. Using only sequence data from L. pneumophila cooling tower isolates and including all core genome variation, we built a multivariate model using discriminant analysis of principal components (DAPC) to find cooling tower-specific genomic signatures, and then used it to predict the origin of clinical isolates. Model assignments were 93% congruent with epidemiological data, including the aquarium Legionnaires outbreak and three other unrelated outbreak investigations. We applied the same approach to a recently described investigation of Legionnaires disease within a UK hospital and observed model predictive ability of 86%. We have developed a promising means to breach L. pneumophila genetic diversity extremes and provide objective source attribution data for outbreak investigations.

microbiology↗

bioRxiv2017

Enterococcus faecalis Promotes Innate Immune Suppression And Polymicrobial Catheter-Associated Urinary Tract Infection

Enterococcus faecalis, a member of the human gastrointestinal microbiota, is an opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. E. faecalis can subvert or evade immune-mediated clearance, although the mechanisms are poorly understood. In this study, we examined E. faecalis-mediated subversion of macrophage activation. We observed that E. faecalis actively prevents NF-{kappa}B signaling in mouse RAW264.7 macrophages in the presence of Toll-like receptor agonists and during polymicrobial infection with Escherichia coli. E. faecalis and E. coli co-infection in a mouse model of catheter-associated urinary tract infection (CAUTI) resulted in a suppressed macrophage transcriptional response in the bladder compared to E. coli infection alone. Finally, we demonstrated that co-inoculation of E. faecalis with E. coli into catheterized bladders significantly augmented E. coli CAUTI. Taken together, these results support that E. faecalis suppression of NF-{kappa}B-driven responses in macrophages promotes polymicrobial CAUTI pathogenesis.\n\nAuthor SummarySynergistic polymicrobial infections can contribute to both disease severity and persistence. Enterococcus faecalis and Escherichia coli are frequently co-isolated from polymicrobial urinary tract infections. Immunomodulation by co-infecting microbes can result in a more permissive environment for pathogens to establish infection. Presently, we do not yet understand how these microbes overcome host immunity to establish polymicrobial infections. To address this, we investigated how the immunosuppressive function of E. faecalis can contribute to acute infection. We defined that E. faecalis is able to suppress macrophages in vitro, despite the presence of E. coli. We also demonstrated E. faecalis ability to augment E. coli titers in vivo to establish kidney infection. Our findings raise the prospect that E. faecalis can alter host immunity to increase susceptibility to other uropathogens.

microbiology↗

bioRxiv2017

Redox-Dependent Condensation Of Mycobacterial Genome By WhiB4

Oxidative stress response in bacteria is generally mediated through coordination between the regulators of oxidant-remediation systems (e.g. OxyR, SoxR) and nucleoid condensation (e.g. Dps, Fis). However, these genetic factors are either absent or rendered nonfunctional in the human pathogen Mycobacterium tuberculosis (Mtb). Therefore, how Mtb organizes genome architecture and regulates gene expression to counterbalance oxidative imbalance during infection is not known. Here, we report that an intracellular redox-sensor, WhiB4, dynamically links genome condensation and oxidative stress response in Mtb. Disruption of WhiB4 affects the expression of genes involved in maintaining redox homeostasis, central carbon metabolism (CCM), respiration, cell wall biogenesis, DNA repair and protein quality control under oxidative stress. Notably, disulfide-linked oligomerization of WhiB4 in response to oxidative stress activates the proteins ability to condense DNA in vitro and in vivo. Further, overexpression of WhiB4 led to hypercondensation of nucleoids, redox imbalance and increased susceptibility to oxidative stress, whereas WhiB4 disruption reversed this effect. In accordance with the findings in vitro, ChIP-Seq data demonstrated non-specific binding of WhiB4 to GC-rich regions of the Mtb genome. Lastly, data indicate that WhiB4 deletion affected the expression of only a fraction of genes preferentially bound by the protein, suggesting its indirect effect on gene expression. We propose that WhiB4 is a novel redox-dependent nucleoid condensing protein that structurally couples Mtbs response to oxidative stress with genome organization and transcription.\n\nSignificance StatementMycobacterium tuberculosis (Mtb) needs to adapt in response to oxidative stress encountered inside human phagocytes. In other bacteria, condensation state of nucleoids modulates gene expression to coordinate oxidative stress response. However, this relation remains elusive in Mtb. We performed molecular dissection of a mechanism controlled by an intracellular redox sensor, WhiB4, in organizing both chromosomal structure and selective expression of adaptive traits to counter oxidative stress in Mtb. Using high-resolution sequencing, transcriptomics, imaging, and redox biosensor, we describe how WhiB4 modulates nucleoid condensation, global gene expression, and redox-homeostasis. WhiB4 over-expression hypercondensed nucleoids and perturbed redox homeostasis whereas WhiB4 disruption had an opposite effect. Our study discovered an empirical role for WhiB4 in integrating redox signals with nucleoid condensation in Mtb.

microbiology↗