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bioRxiv2017

Prey range and genome evolution of Halobacteriovorax marinus predatory bacteria from an estuary

BackgroundHalobacteriovorax are saltwater-adapted predatory bacteria that attack Gram-negative bacteria and therefore may play an important role in shaping microbial communities. To understand the impact of Halobacteriovorax on ecosystems and develop them as biocontrol agents, it is important to characterize variation in predation phenotypes such as prey range and investigate the forces impacting Halobacteriovorax genome evolution across different phylogenetic distances.\n\nResultsWe isolated H. marinus BE01 from an estuary in Rhode Island using Vibrio from the same site as prey. Small, fast-moving attack phase BE01 cells attach to and invade prey cells, consistent with the intraperiplasmic predation strategy of H. marinus type strain SJ. BE01 is a prey generalist, forming plaques on Vibrio strains from the estuary as well as Pseudomonas from soil and E. coli. Genome analysis revealed that BE01 is very closely related to SJ, with extremely high conservation of gene order and amino acid sequences. Despite this similarity, we identified two regions of gene content difference that likely resulted from horizontal gene transfer. Analysis of modal codon usage frequencies supports the hypothesis that these regions were acquired from bacteria with different codon usage biases compared to Halobacteriovorax. In BE01, one of these regions includes genes associated with mobile genetic elements, such as a transposase not found in SJ and degraded remnants of an integrase occurring as a full-length gene in SJ. The corresponding region in SJ included unique mobile genetic element genes, such as a site-specific recombinase and bacteriophage-related genes not found in BE01. Acquired functions in BE01 include the dnd operon, which encodes a pathway for DNA modification that may protect DNA from nucleases, and a suite of genes involved in membrane synthesis and regulation of gene expression that was likely acquired from another Halobacteriovorax lineage.\n\nConclusionsOur results support previous observations that Halobacteriovorax prey on a broad range of Gram-negative bacteria. Genome analysis suggests strong selective pressure to maintain the genome in the H. marinus lineage represented by BE01 and SJ, although our results also provide further evidence that horizontal gene transfer plays an important role in genome evolution in predatory bacteria.

microbiology↗

bioRxiv2017

The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A Carbohydrate in Streptococcus pyogenes

In many Lactobacillales species (i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes, synthesizes a key antigenic surface polymer--the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N-acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphorylundecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltranferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis. In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.

microbiology↗

bioRxiv2017

Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1

Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate (NO3-) to nitrite (NO2-), which accumulates in the medium. These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (NarGHJI). M. nitratireducenticrescens strain JAM1 also contains gene clusters encoding two putative nitric oxide reductase (NO) reductases and one putative nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. In this study, we show that strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory copper-(NirK) or cytochrome cd1-type (NirS) NO2- reductase, NO3--amended strain JAM1 cultures produce N2O, representing up to 6% of the N-input. NO2- was shown to be the key intermediate of this production process. In NO3-amended cultures, we analyzed denitrification genes in succession of net N2O-production and -consumption phases at the gene expression level. These phases were found to correlate with changes in the expression levels of the NO reductase gene cnorB1 and nnrS, which indicated NO production in the cultures.\n\nImportanceBy showing that all the three denitrification reductases are active, this demonstrates that Methylophaga nitratireducenticrescens JAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of a denitrification reactor from which it originates. The small production of N2O via NO in strain JAM1 did not involve Nar contrary to what was demonstrated in Escherichia coli. M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using NO3- and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities.

microbiology↗

bioRxiv2017

Super-Resolution Microscopy Reveals Structural Mechanisms Driving the Nanoarchitecture of a Viral Chromatin Tether

By tethering their circular genomes (episomes) to host chromatin, DNA tumor viruses ensure retention and segregation of their genetic material during cell divisions. Despite functional genetic and crystallographic studies, there is little information addressing the three-dimensional structure of these tethers in cells, issues critical for understanding persistent infection by these viruses. Here, we have applied direct stochastic optical reconstruction microscopy (dSTORM) to establish the nanoarchitecture of tethers within cells latently infected with the oncogenic human pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV). Each KSHV tether comprises a series of homodimers of the latency-associated nuclear antigen (LANA) that bind with their C-termini to the tandem array of episomal terminal repeats (TRs) and with their N-termini to host chromatin. Super-resolution imaging revealed that individual KSHV tethers possess similar overall dimensions and, in aggregate, fold to occupy the volume of a prolate ellipsoid. Using plasmids with increasing numbers of TRs, we found that tethers display polymer power-law scaling behavior with a scaling exponent characteristic of active chromatin. For plasmids containing a two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corresponding to a single TR. From these data, we have generated a three-dimensional model of the episomal half of the tether that integrates and extends previously established findings from epi-fluorescent, crystallographic, and epigenetic approaches. Our findings also validate the use of dSTORM in establishing novel structural insights into the physical basis of molecular connections linking host and pathogen genomes.

microbiology↗

bioRxiv2017

Plant genes influence microbial hubs that shape beneficial leaf communities

Although complex interactions between hosts and microbial associates are increasingly well documented, we still know little about how and why hosts shape microbial communities in nature. In addition, host genetic effects on microbial communities vary widely depending on the environment, obscuring conclusions about which microbes are impacted and which plant functions are important. We characterized the leaf microbiota of 200 A. thaliana genotypes in eight field experiments and detected consistent host effects on specific, broadly distributed microbial OTUs. Host genetics disproportionately influenced hubs within the microbial communities, with their impact then percolating through the community, as evidenced by a decline in the heritability of particular OTUs with their distance to the nearest hub. By simultaneously measuring host performance, we found that host genetics associated with microbial hubs explained over 10% of the variation in lifetime seed production among host genotypes across sites and years. We successfully cultured one of these microbial hubs and demonstrated its growth-promoting effects on plants grown in sterile conditions. Finally, genome-wide association mapping identified many putatively causal genes with small effects on the relative abundance of microbial hubs across sites and years, and these genes were enriched for those involved in the synthesis of specialized metabolites, auxins and the immune system. Using untargeted metabolomics, we corroborate the consistent association of variation in specialized metabolites and microbial hubs across field sites. Together, our results reveal that host natural variation impacts the microbial communities in consistent ways across environments and that these effects contribute to fitness variation among host genotypes.

microbiology↗

bioRxiv2017

Genomic and proteomic analysis of Human herpesvirus 6 reveals distinct clustering of acute versus inherited forms and reannotation of reference strain

Human herpesvirus-6A and -6B (HHV-6) are betaherpesviruses that reach >90% seroprevalence in the adult population. Unique among human herpesviruses, HHV-6 can integrate into the subtelomeric regions of human chromosomes; when this occurs in germ line cells it causes a condition called inherited chromosomally integrated HHV-6 (iciHHV-6). To date, only two complete genomes are available for HHV-6B. Using a custom capture panel for HHV-6B, we report near-complete genomes from 61 isolates of HHV-6B from active infections (20 from Japan, 35 from New York state, and 6 from Uganda), and 64 strains of iciHHV-6B (mostly from North America). We also report partial genome sequences from 10 strains of iciHHV-6A. Although the overall sequence diversity of HHV-6 is limited relative to other human herpesviruses, our sequencing identified geographical clustering of HHV-6B sequences from active infections, as well as evidence of recombination among HHV-6B strains. One strain of active HHV-6B was more divergent than any other HHV-6B previously sequenced. In contrast to the active infections, sequences from iciHHV-6 cases showed reduced sequence diversity. Strikingly, multiple iciHHV-6B sequences from unrelated individuals were found to be completely identical, consistent with a founder effect. However, several iciHHV-6B strains intermingled with strains from active pediatric infection, consistent with the hypothesis that intermittent de novo integration into host germline cells can occur during active infection Comparative genomic analysis of the newly sequenced strains revealed numerous instances where conflicting annotations between the two existing reference genomes could be resolved. Combining these findings with transcriptome sequencing and shotgun proteomics, we reannotated the HHV-6B genome and found multiple instances of novel splicing and genes that hitherto had gone unannotated. The results presented here constitute a significant genomic resource for future studies on the detection, diversity, and control of HHV-6.\n\nAuthor SummaryHHV-6 is a ubiquitous large DNA virus that is the most common cause of febrile seizures and reactivates in allogeneic stem cell patients. It also has the unique ability among human herpesviruses to be integrated into the genome of every cell via integration in the germ line, a condition called inherited chromosomally integrated (ici)HHV-6, which affects approximately 1% of the population. To date, very little is known about the comparative genomics of HHV-6. We sequenced 61 isolates of HHV-6B from active infections, 64 strains of iciHHV-6B, and 10 strains of iciHHV-6A. We found geographic clustering of HHV-6B strains from active infections. In contrast, iciHHV-6B had reduced sequence diversity, with many identical sequences of iciHHV-6 found in individuals not known to share recent common ancestry, consistent with a founder effect from a remote common ancestor with iciHHV-6. We also combined our genomic analysis with transcriptome sequencing and shotgun proteomics to correct previous misannotations of the HHV-6 genome.

microbiology↗

bioRxiv2017

Fast and Simple Tool for the Quantification of Biofilm-Embedded Cells Sub-Populations From Fluorescent Microscopic Images

Fluorescent staining is a common tool for both quantitative and qualitative assessment of pro- and eukaryotic cells sub-population fractions by using microscopy and flow cytometry. However, direct cell counting by flow cytometry is often limited, for example when working with cells rigidly adhered either to each other or to external surfaces like bacterial biofilms or adherent cell lines and tissue samples. An alternative approach is provided by using fluorescent microscopy and confocal laser scanning microscopy (CLSM), which enables the evaluation of fractions of cells subpopulations in a given sample. For the quantitative assessment of cell fractions in microphotographs, we suggest a simple two-step algorithm that combines single cells selection and the statistical analysis. To facilitate the first step, we suggest a simple procedure that supports finding the balance between the detection threshold and the typical size of single cells based on objective cell size distribution analysis. Based on a series of experimental measurements performed on bacterial and eukaryotic cells under various conditions, we show explicitly that the suggested approach effectively accounts for the fractions of different cell sub-populations (like the live/dead staining in our samples) in all studied cases that are in good agreement with manual cell counting on microphotographs and flow cytometry data. This algorithm is implemented as a simple software tool that includes an intuitive and user-friendly graphical interface for the initial adjustment of algorithm parameters to the microphotographs analysis as well as for the sequential analysis of homogeneous series of similar microscopic images without further user intervention. The software tool entitled BioFilmAnalyzer is freely available online at https://bitbucket.org/rogex/biofilmanalyzer/downloads/.

microbiology↗

bioRxiv2017

Dominance of ST131 Escherichia coli carrying blaCTX-M in patients with bloodstream infections caused by cephalosporin-resistant strains in Australia, New Zealand and Singapore: whole genome analysis of isolates from a randomised trial

Synopsis/AbstractO_ST_ABSObjectivesC_ST_ABSTo characterise multi-drug resistant Escherichia coli isolated from patients in Australia, New Zealand and Singapore with bloodstream infection (BSI).\n\nMethodsWe prospectively collected third-generation cephalosporin resistant (3GC-R) E. coli from blood cultures obtained from patients enrolled in a randomised controlled trial. Whole genome sequencing was used to characterise antibiotic resistance genes, sequence types (STs), plasmids and phylogenetic relationships. Antibiotic susceptibility was determined using disk diffusion and Etest.\n\nResultsA total of 70 E. coli were included, of which the majority were ST131 (61.4%). BSI was most frequently from a urinary source (69.6%), community-associated (62.9%) and in older patients (median age 71 years [IQR 64-81]). The median Pitt bacteraemia score at presentation was 1 (IQR 0-2, range 0-3) and ICU admission was infrequent (3.1%). ST131 possessed significantly more acquired resistance genes than non-ST131 (p=0.003). Clade C1/C2 ST131 predominated (30.2% and 53.5% of all ST131 respectively) and these were all resistant to ciprofloxacin. All clade A ST131 were community-associated. The predominant ESBL types were blaCTX-M (78.6% of isolates) and were strongly associated with ST131, with the majority blaCTX-M-15. Clade C1 was associated with blaCTX-M-14 and blaCTX-M-27, whereas blaCTX-M-15 predominated in clade C2. Plasmid-mediated AmpC (p-AmpC) genes (mainly blaCMY-2) were also frequent (17.1%) but were more common with non-ST131 strains (p< 0.001). The majority of plasmid replicon types were IncF.\n\nConclusionsIn a prospective collection of 3GC-R E. coli causing BSI in the Australasian region, community-associated Clade C1/C2 ST131 predominate in association with blaCTX-M ESBLs, although a significant proportion of non-ST131 strains carried blaCMY-2.

microbiology↗

bioRxiv2017

Anaerobic methanotrophic communities thrive in deep submarine permafrost

Thawing submarine permafrost is a source of methane to the subsurface biosphere. Methane oxidation in submarine permafrost sediments has been proposed, but the responsible microorganisms remain uncharacterized. We analyzed archaeal communities and identified distinct anaerobic methanotrophic (ANME-2a/b, ANME-2d) assemblages in frozen and completely thawed submarine permafrost sediments. Besides archaea potentially involved in AOM we found a large diversity of archaea mainly belonging to Bathyarchaeota, Thaumarchaeota, and Euryarchaeota. Methane concentrations and {delta}13C-methane signatures distinguish horizons of potential anaerobic oxidation of methane (AOM) coupled either to sulfate reduction in a sulfate-methane transition zone (SMTZ) or to the reduction of other electron acceptors, such as iron, manganese or nitrate. Analysis of functional marker genes (mcrA) and fluorescence in situ hybridization (FISH) corroborate AOM communities in submarine permafrost sediments potentially active at low temperatures. Extrapolating potential AOM rates, when scaled to the total area of expected submarine permafrost thaw, reveals that methane could be consumed at rates between 8 and 120 Tg C per year, which is comparable to other AOM habitats such as seeps, continental SMTZ and wetlands. We thus propose that AOM is active where submarine permafrost thaws and needs to be accounted for in global methane budgets.

microbiology↗

bioRxiv2017

Mouse hepatitis virus nsp14 exoribonuclease activity is required for resistance to innate immunity

ABSTRACTCoronaviruses (CoV) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that interact with the host innate immune response to facilitate efficient viral replication. CoV non-structural protein 14 (nsp14) encodes 3'-to-5' exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order Nidovirales, arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade dsRNA replication intermediates. In this study, we tested the hypothesis that CoV ExoN may also function to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-{beta}) in a dose-dependent manner. In addition, ExoN(-) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor deficient (IFNAR-/-) BMMs. ExoN(-) virus replication did not result in IFN-{beta} gene expression, and in the presence of an IFN-{beta}-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to untreated. However, ExoN(-) virus generated from IFN-{beta} pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest MHV ExoN activity is required for resistance to the innate immune response and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity.

microbiology↗

bioRxiv2017

Ecophylogenetics Reveals the Evolutionary Associations between Mammals and their Gut Microbiota

A tantalizing hypothesis posits that mammals coevolved with their gut microbiota. Unfortunately, the limited resolution of microbial taxonomy hinders the exploration of this hypothesis and specifically challenges the discovery of gut microbes that are linked to mammalian evolution. To address this, we developed a novel approach that groups microbes into new, more meaningful taxonomic units based on their common ancestry and ecological redundancy. Treating mammalian lineages as different ecosystems, we quantified the distribution of these microbial taxa across mammals. Our analysis discovered monophyletic clades of gut bacteria that are unexpectedly prevalent, or conserved, across all mammals, as well as conserved clades that are exclusive to particular mammalian lineages. These clades often manifest phylogenetic patterns indicating that they are subject to selection. Lineage - specific changes in clade conservation, including a human-accelerated loss of conserved clades, suggest that mammalian evolution associates with a change in the selective regimes that act on gut microbiota. Collectively, these results point to the existence of microbes that possess traits that facilitate their dispersion or survival in the mammalian gut, possibly because they are subject to host selection. Ultimately, our analysis clarifies the relationship between the diversification of the gut microbiome and mammalian evolutionary history.

microbiology↗

bioRxiv2017

HPeV-3 predominated among Parechovirus A positive infants in the summer of 2013-2014 in Queensland, Australia.

Parechoviruses (HPeV) are not new viruses and are found in the respiratory tract and central nervous system of children and adults in conjunction with a range of acute illnesses. During an Australian outbreak of HPeV in the summer of 2013, we performed PCR-based screening and genotyping to determine whether ill Queensland infants were infected by HPeV. HPeVs were detected among 25/62 samples, identified as HPeV-3 from 23 that could be genotyped. These variants closely matched those occurring during and after the 2013 HPeV season. The inclusion of HPeV screening should be considered among acutely ill young infants during summer.\n\nHighlightsO_LIHPeV-3 was the most common Parechovirus A genotype in Queensland summer of 2013/14\nC_LIO_LIHPeV testing should be routine among testing of infants with acute CNS - related symptoms\nC_LIO_LIHPeV is a seasonal virus\nC_LIO_LISubgenomic phylogenetic analysis of HPeVs can be confounded by the presence of recombination\nC_LI

microbiology↗

bioRxiv2017

Synergy of Quorum Quenching Enzyme and Quorum Sensing Inhibitor in Inhibiting P. aeruginosa Quorum Sensing

The threat of antibiotic resistant bacteria has called for alternative antimicrobial strategies that would mitigate the increase of classical resistance mechanism. Many bacteria employ quorum sensing (QS) to govern the production of virulence genes and formation of drug-resistance biofilms. Blocking QS mechanisms have proven to be a functional alternative to conventional antibiotic control of infections. The concepts of quorum sensing inhibitors (QSI) and quorum quenching enzymes (QQ) have been investigated separately. In this study however, we simulated the synergistic effect of QQ and QSI in blocking bacterial QS. This effect was validated by experiments using AiiA and G1 as QQ and QSI respectively on Pseudomonas aeruginosa LasR/I and RhlR/I QS circuits. The combination of a QQ and a QSI almost completely blocked the P. aeruginosa QS las and rhl system. Our findings provided a potential application strategy for bacterial QS disruption.

microbiology↗

bioRxiv2017

Streptolysin-induced endoplasmic reticulum stress promotes group A streptococcal in vivo biofilm formation and necrotizing fasciitis

Group A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotizing fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions and the host-pathogen interactions that might influence biofilm formation. Here we establish and characterize an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a murine model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a three-dimensional architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) resulting in the release of a host-associated biofilm promoting-factor(s). Supernatants from GAS-infected mammalian cells or from cells treated with endoplasmic reticulum (ER) stressors restore biofilm formation to an SLO and SLS null mutant that is otherwise attenuated in biofilm formation on cells, together suggesting a role for streptolysin-induced ER stress in this process. In an in vivo mouse model, the streptolysin-null mutant is attenuated in both microcolony formation and bacterial spread, but pre-treatment of softtissue with an ER-stressor restores the ability of the mutant to form wild type like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression.\n\nSignificance StatementAlthough it is well-accepted that bacterial biofilms are associated with many chronic infections, little is known about the mechanisms by which group A Streptococcus (GAS) biofilms contribute to acute soft tissue-invasive diseases like necrotizing fasciitis (NF). In this study, we establish a physiologically relevant in vitro model to study GAS biofilm formation on mammalian cells and validate our findings in a mouse model that mimics human NF. This study demonstrates a novel role of GAS streptolysin-mediated ER stress in the development and spread of GAS biofilms in acute softtissue infections. We also show that biofilm formation depends on the release of a host-associated factor that promotes microcolony formation and GAS dissemination in vivo.

microbiology↗

bioRxiv2017

Validation of RT-qPCR approaches to monitor Pseudomonas syringae gene expression during infection and exposure to pattern-triggered immunity

Pseudomonas syringae pv. tomato DC3000 (DC3000) is an important model plant pathogen, with a fully annotated genome and multiple compatible plant hosts. Very few studies have examined the regulation of DC3000 gene expression in vivo. We developed a RT-qPCR assay to monitor transcriptional changes in DC3000 inoculated into Arabidopsis thaliana leaves during disease and exposure to pattern-triggered immunity (PTI). In our approach, bacterial RNA concentrations in total tissue RNA are standardized using P.syringae-specific16S ribosomal RNA primers. We validated multiple stable reference genes for normalization in calculating the relative expression of genes of interest. We used empirically derived rates of amplification efficiency to calculate relative expression of key marker genes for virulence-associated regulation. We demonstrated that exposure to PTI alters DC3000 expression of Type III secretion system, coronatine synthesis genes and flagellar marker genes.

microbiology↗

bioRxiv2017

Nutrient stoichiometry shapes microbial coevolution

Coevolution is a force contributing to the generation and maintenance of biodiversity. It is influenced by environmental conditions including the scarcity of essential resources, which can drive the evolution of defense and virulence traits. We conducted a long-term chemostat experiment where the marine cyanobacterium Synechococcus was challenged with a lytic phage under nitrogen (N) or phosphorus (P) limitation. This manipulation of nutrient stoichiometry altered the stability of host-parasite interactions and the underlying mode of coevolution. By assessing infectivity with >18,000 pairwise challenges, we documented directional selection for increased phage resistance, consistent with arms-race dynamics while phage infectivity fluctuated through time, as expected when coevolution is driven by negative frequency-dependent selection. The resulting infection networks were 50 % less modular under N-versus P-limitation reflecting host-range contraction and asymmetric coevolutionary trajectories. Nutrient stoichiometry affects eco-evolutionary feedbacks in ways that may alter the dynamics and functioning of environmental and host-associated microbial communities.

microbiology↗

bioRxiv2017

Dual RNAseq shows the human mucosal immunity protein, MUC13, is a hallmark of Plasmodium exoerythrocytic infection

The exoerythrocytic stage of Plasmodium malaria infection is a critical window for prophylactic intervention. Using a genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we identify the human mucosal immunity gene, Mucin13 (MUC13), as strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm that MUC13 expression is upregulated in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, distinguishing both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes across all Human-infecting Plasmodium species. This data presents a novel interface of host-parasite interactions in Plasmodium, in that a component of host mucosal immunity is reprogrammed to assist the progression of infection.

microbiology↗

bioRxiv2017

The ABCE1 capsid assembly pathway is conserved between primate lentiviruses and the non-primate lentivirus feline immunodeficiency virus

During immature capsid assembly in cells, the Gag protein of HIV-1 and other primate lentiviruses co-opts a host RNA granule, forming a pathway of assembly intermediates that contains host components, including two cellular enzymes shown to facilitate assembly, ABCE1 and DDX6. Here we asked whether a non-primate lentivirus, feline immunodeficiency virus (FIV), also forms such RNA-granule-derived intracellular capsid assembly intermediates. First, we found that, unlike for HIV-1, the FIV completed immature capsid and the largest putative assembly intermediate are unstable during analysis. Next, we identified in situ cross-linking conditions that overcame this problem and revealed the presence of FIV Gag complexes that correspond in size to early and late HIV-1 assembly intermediates. Because assembly-defective HIV-1 Gag mutants are arrested at specific intracellular assembly intermediates, we asked if a similar arrest is also observed for FIV. We analyzed four FIV Gag mutants, including three not previously studied that we identified based on sequence and structural similarity to HIV-1 Gag, and found that each is assembly-defective and arrested at the same intermediate as the corresponding HIV-1 mutant. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from co-immunoprecipitation studies demonstrating that FIV Gag is associated with ABCE1 and DDX6, as shown previously for HIV-1. Finally, we validated these co-immunoprecipitations with a proximity ligation assay that revealed co-localization between assembly-competent FIV Gag and ABCE1 in situ. Together, these data offer novel structure-function insights and indicate that primate and non-primate lentiviruses form intracellular capsid assembly intermediates derived from ABCE1-containing RNA granules.\n\nImportanceLike HIV-1, FIV Gag assembles into immature capsids; however, it is not known whether FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we asked whether FIV Gag forms complexes similar in size to HIV-1 assembly intermediates and if FIV Gag is associated with ABCE1 and DDX6, two host enzymes that facilitate HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies identified FIV Gag-containing complexes that closely resemble HIV-1 capsid assembly intermediates, showed that known and novel assembly-defective FIV Gag mutants fail to progress past these putative intermediates, and utilized biochemical and imaging approaches to demonstrate association of FIV Gag with ABCE1 and DDX6. Thus, we conclude that viral-host interactions important for immature capsid assembly are conserved between primate and non-primate lentiviruses, and could yield important targets for future antiviral strategies.

microbiology↗