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bioRxiv2018

The Pseudomonas aeruginosa LasR quorum-sensing receptor balances ligand selectivity and sensitivity

Quorum sensing is a cell-cell communication process that bacteria use to orchestrate group behaviors. Quorum sensing is mediated by extracellular signal molecules called autoinducers. Autoinducers are often structurally similar, raising questions concerning how bacteria distinguish among them. Here, we use the Pseudomonas aeruginosa LasR quorum-sensing receptor to explore receptor sensitivity and selectivity. Alteration of LasR amino acid S129 increases ligand selectivity and decreases ligand sensitivity. Conversely, the L130F mutation enhances LasR sensitivity while reducing selectivity. We solve crystal structures of LasR ligand binding domains complexed with non-cognate autoinducers. Comparison to existing structures reveals that ligand selectivity/sensitivity is mediated by a flexible loop adjacent to the ligand binding site. We show that P. aeruginosa harboring LasR variants with modified selectivity or sensitivity exhibit altered quorum-sensing responses. We suggest that an evolutionary trade-off between ligand selectivity and sensitivity enables LasR to optimally regulate quorum-sensing traits.

microbiology↗

bioRxiv2018

Probing host-anaerobe interactions in innovative human gut cellular models

Interactions of anaerobic gut bacteria, such as Clostridium difficile, with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Although gut colonization by C. difficile is a key determinant of disease outcome, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut epithelial monolayers co-cultured within dual environment chambers, we demonstrate that C. difficile adhesion to gut epithelial cells is accompanied by a gradual increase in bacterial numbers. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, toxins and bacterial filaments. This 2-D dual chamber system was used to examine C. difficile interactions with the commensal Bacteroides dorei, and interestingly, C. difficile growth is significantly reduced in presence of B. dorei. Furthermore, in novel multilayer and 3-D gut models containing a myofibroblast layer, C. difficile adheres more efficiently to epithelial cells, as compared to the 2-D model, leading to a quicker destruction of the epithelium. Our study describes new controlled environment human gut models that enable host-anaerobe and pathogen-commensal interaction studies in vitro.

microbiology↗

bioRxiv2018

Rifampicin or capreomycin induced remodelling of the Mycobacterium smegmatis mycolic acid layer is mitigated in synergistic combinations with cationic antimicrobial peptides

The mycobacterial cell wall affords natural resistance to antibiotics. Antimicrobial peptides (AMPs) modify the surface properties of mycobacteria and can act synergistically with antibiotics from differing classes. Here we investigate the response of Mycobacterium smegmatis to the presence of rifampicin or capreomycin, either alone or in combination with two synthetic, cationic, -helical AMPs; distinguished by the presence (D-LAK120-HP13) or absence (D-LAK120-A) of a kink-inducing proline. Using a combination of high-resolution magic angle spinning (HR-MAS) NMR of bacteria, diphenylhexatriene (DPH) fluorescence anisotropy and laurdan emission spectroscopy we show that M. smegmatis responds to challenge with rifampicin or capreomycin by substantially altering its metabolism and, in particular, by remodelling the cell envelope. In NMR spectra of bacteria, reductions in intensity for mycolic acid lipid -(CH2)-, -CH3, R2CH-COOH, R2CH-OH and also -CH2-(CH==CH)- and -CH=CH- resonances were observed following challenge with rifampicin and capreomycin, while the latter also caused an increase in trehalose. These changes are consistent with a reduction of trehalose dimycolate and increase of trehalose monomycolate and are associated with an increase in rigidity of the mycolic acid layer observed following challenge by capreomycin but not rifampicin. Challenge with D-LAK120-A or D-LAK120-HP13 induced no or modest changes respectively in these metabolites and did not induce a significant increase in rigidity of the mycolic acid layer. Further, the response to rifampicin or capreomycin was significantly reduced when these were combined respectively with D-LAK120-HP13 and D-LAK120-A, suggesting a possible mechanism for the synergy of these combinations. The remodelling of the mycomembrane in M. smegmatis is therefore identified as an important countermeasure deployed against rifampicin or capreomycin, but this can be mitigated, and rifampicin or capreomycin efficacy potentiated, by combining with AMPs.

microbiology↗

bioRxiv2018

Structural diversity across arbuscular mycorrhizal, ectomycorrhizal, and endophytic plant-fungus networks

BackgroundBelow-ground linkage between plant and fungal communities is one of the major drivers of terrestrial ecosystem dynamics. However, we still have limited knowledge of how such plant-fungus associations vary in their community-scale properties depending on fungal functional groups and geographic locations.\n\nMethodsBased on high-throughput sequencing of root-associated fungi in eight forests along the Japanese Archipelago, we performed a comparative analysis of arbuscular mycorrhizal, ectomycorrhizal, and saprotrophic/endophytic associations across a latitudinal gradient from cool-temperate to subtropical regions.\n\nResultsIn most of the plant-fungus networks analyzed, host-symbiont associations were significantly specialized but lacked \"nested\" architecture, which has been commonly reported in plant-pollinator and plant-seed disperser networks. Meanwhile, the structure of arbuscular mycorrhizal networks was differentiated from that of ectomycorrhizal and saprotrophic/endophytic networks, characterized by high connectance. Our data also suggested that geographic factors affected the organization of plant-fungus network structure. For example, the southernmost subtropical site analyzed in this study displayed lower network-level specificity of host-symbiont associations and higher (but still low) nestedness than northern localities.\n\nConclusionsOur comparative analyses suggest that arbuscular mycorrhizal, ectomycorrhizal, and saprotrophic/endophytic plant-fungus associations often lack nested network architecture, while those associations can vary, to some extent, in their community-scale properties along a latitudinal gradient. Overall, this study provides a basis for future studies that will examine how different types of plant-fungus associations collectively structure terrestrial ecosystems.

microbiology↗

bioRxiv2018

Soil microbial habitats in an extreme desert Mars-analogue environment

Sediments in the hyper-arid core of the Atacama Desert are a terrestrial analogue to Mars regolith. Understanding the distribution and drivers of microbial life in the sediment may give critical clues on how to search for biosignatures on Mars. Here, we identify the spatial distribution of highly specialised bacterial communities in previously unexplored depth horizons of subsurface sediments. We deployed an autonomous rover in a mission-relevant Martian drilling scenario with manual sample validation. Subsurface communities were delineated by depth related to sediment moisture. Geochemical analysis indicated soluble salts and minerology that influenced water bio-availability, particularly in deeper sediments. Colonization was also patchy and uncolonized sediment was associated with indicators of extreme osmotic challenge. The study identifies linkage between biocomplexity, moisture and geochemistry in Mars-like sediments at the limit of habitability and demonstrates feasibility of the rover-mounted drill for future Mars sample recovery.

microbiology↗

bioRxiv2018

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human group IIA secreted phospholipase A2 to exert its bactericidal effects

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant human hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the{Delta} gacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the human bactericidal enzyme hGIIA to exert its bactericidal function.\n\nAuthor summaryThe human immune system is capable of killing invading bacteria by secreting antimicrobial proteins. Cationic human Group IIA secreted phospholipase A2 (hGIIA) is especially effective against Gram-positive bacteria by degrading the bacterial membrane. HGIIA requires binding to negatively charged surface structures before it can penetrate through the thick peptidoglycan layer and reach the target phospholipid membrane. HGIIA is constitutively expressed at high concentrations at sites of possible bacterial entry, e.g. in tears, skin and small intestine. In serum, normal concentrations are low but can increase up to 1,000-fold upon inflammation or infection. In vitro, ex vivo and in vivo experiments suggest an important role for hGIIA in defense against two human pathogens, Group A and Group B Streptococcus (GAS, GBS). We demonstrate that the Lancefield cell wall polysaccharides that are expressed by these bacteria, the Group A Carbohydrate (GAC) for GAS and the Group B Carbohydrate (GBC) for GBS, are required for optimal hGIIA bactericidal efficacy by facilitating penetration through the peptidoglycan layer. Given the increased hGIIA resistance of antigen-modified or antigen-deficient streptococci, it will be of interest to determine potential regulatory mechanisms regarding expression of streptococcal Lancefield polysaccharides.

microbiology↗

bioRxiv2018

Microbiomes of North American Triatominae: the grounds for Chagas disease epidemiology

AbstarctInsect microbiomes influence many fundamental host traits, including functions of practical significance such as their capacity as vectors to transmit parasites and pathogens. The knowledge on the diversity and development of the gut microbiomes in various blood feeding insects is thus crucial not only for theoretical purposes, but also for the development of better disease control strategies. In Triatominae (Heteroptera: Reduviidae), the blood feeding vectors of Chagas disease in South America and parts of North America, the investigation of the microbiomes is in its infancy. The few studies done on microbiomes of South American Triatominae species indicate a relatively low taxonomic diversity and a high host specificity. We designed a comparative survey to serve several purposes: I) to obtain a better insight into the overall microbiome diversity in different species, II) to check the long term stability of the interspecific differences, III) to describe the ontogenetic changes of the microbiome, and IV) to determine the potential correlation between microbiome composition and presence in the vector gut of Trypanosoma cruzi, the causative agent of Chagas disease. Using 16S amplicons of two abundant species from the southern US, and four laboratory reared colonies, we showed that the microbiome composition is determined by host species, rather than locality or environment. The OTUs (Operational Taxonomic Units) determination confirms a low microbiome diversity, with 12-17 main OTUs detected in wild populations of T. sanguisuga and T. protracta. Among the dominant bacterial taxa are Acinetobacter and Proteiniphilum but also the symbiotic bacterium Arsenophonus triatominarum, previously believed to only live intracellularly. The possibility of ontogenic microbiome changes was evaluated in all six developmental stages and faeces of the laboratory reared model Rhodnius prolixus. We detected considerable changes along the hosts ontogeny, including clear trends in the abundance variation of the three dominant bacteria, namely Enterococcus, Acinetobacter and Arsenophonus. Finally, we screened the samples for the presence of T. cruzi. Comparing the parasite presence with the microbiome composition, we assessed the possible significance of the latter in the epidemiology of the disease. Particularly, we found a trend towards more diverse microbiomes in T. cruzi positive T. protracta specimens.

microbiology↗

bioRxiv2018

Household Transmission Study of Cryptosporidiosis in Bangladesh

BackgroundCryptosporidium, an apicomplexan protozoa, is a leading contributor to diarrheal morbidity and mortality in children under five years old worldwide. As there is no vaccine and no approved drug for Cryptosporidium spp. in young children, preventing parasite transmission is crucial. We undertook a pilot case-control study to define the extent of person-to-person transmission of cryptosporidiosis within families in an urban and rural community in Bangladesh.\n\nMethodsWe enrolled 48 case families with a Cryptosporidium-infected child aged 6-18 months. Controls were age-sex matched Cryptosporidium-negative children (n=12). Once children were identified, we enrolled all household members. We then followed these individuals for 8 weeks, with weekly surveillance stools and testing with qPCR for Cryptosporidium spp.\n\nFindingsIn the 48 case families, the rate of secondary infections with Cryptosporidium was 18.6% (22/118) compared to 0 new infections (0/35) in the 12 control families. In the 22 urban Mirpur households, the secondary attack rate was 30% (18/60) in cases compared to 0% (0/14) in controls (chi-square p = 0.018). In contrast, in the 21 rural Mirzapur households, the secondary attack rate was 6.9% (4/58) in case households compared to 0% (0/21) in controls (chi-square p = 0.22). Genotyping by gp60 demonstrated infection with the same subspecies in five of six families. Serologic response to Cryptosporidium infection was associated with younger age, longer duration of infection, and C hominis gp60_IbA9G3R2 infection.\n\nInterpretationThe high rate of secondary infection in Mirpur suggests that person-to-person transmission is likely a major source of Cryptosporidium infection for young children living in this region. GP 60 genotyping demonstrated direction of infection in 2 households, and concurrent infection in five households. Further work is needed to understand the differences in parasite transmissibility and immunity to different genotypes.

microbiology↗

bioRxiv2018

The development of gut microbiota in ostriches and its association with growth

The development of gut microbiota during ontogeny in vertebrates is emerging as an important process influencing physiology, immune system, health, and adult fitness. However, we have little knowledge of how the gut microbiome is colonised and develops in non-model organisms, and to what extent microbial diversity and specific taxa influence changes in fitness-related traits. Here, we used 16S rRNA gene sequencing to describe the successional development of the faecal microbiota in juvenile ostriches (Struthio camelus; n = 71) over their first three months of life, during which time a five-fold difference in weight was observed. We found a gradual increase in microbial diversity with age, an overall convergence in community composition among individuals, multiple colonisation and extinction events, and major taxonomic shifts coinciding with the cessation of yolk absorption. In addition, we discovered significant but complex associations between juvenile growth and microbial diversity, and identified distinct bacterial groups that had positive (Bacteroidaceae) and negative (Enterobacteriaceae, Enterococcaceae, Lactobacillaceae) correlations with the growth of individuals at specific ages. These results have broad implications for our understanding of the development of gut microbiota and its association with juvenile growth.

microbiology↗

bioRxiv2018

Tandem tyrosine residues in the EPEC multicargo chaperone CesT support differential type III effector translocation and early host colonization

Enteropathogenic Escherichia coli (EPEC) are worldwide human enteric pathogens inflicting significant morbidity and causing large economic losses. A type 3 secretion system (T3SS) is critical for EPEC intestinal colonization, and injection of effectors into host cells contributes to cellular subversion and innate immune evasion. Here, we demonstrate that two strictly conserved C-terminal tyrosine residues, Y152 and Y153, within the multicargo T3SS chaperone CesT serve differential roles in regulating effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine attenuated EPEC type 3 effector injection into host cells, and significantly limited Tir effector mediated intimate adherence, a key feature of attaching and effacing pathogenesis. Whereas CesT Y153 supported normal levels of Tir translocation, CesT Y152 was strictly required for the effector NleA to be expressed and subsequently translocated into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y152, Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating Y152 in CesT functionality. A mouse infection model of EPEC intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for strictly conserved tandem tyrosine residues within CesT. Tyrosine 152 of CesT is implicated in NleA expression, providing functional relevance for localized amino acid conservation. Therefore, CesT via its novel C-terminal domain, has relevant roles beyond typical T3SC that interact and stabilize effector proteins.

microbiology↗

bioRxiv2018

An Integrin-Independent Kaposi’s Sarcoma-Associated Herpesvirus Infection Mechanism

Host receptor usage by KSHV has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding, then interacts with a complex of EphA2, xct, and integrins 3{beta}1, V{beta}3, V{beta}5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of V- and {beta}1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.\n\nIMPORTANCEOur data reveals an integrin-independent route of KSHV infection and suggests that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.

microbiology↗

bioRxiv2018

Network hubs in root-associated fungal metacommunities

BackgroundAlthough a number of recent studies have uncovered remarkable diversity of microbes associated with plants, understanding and managing dynamics of plant microbiomes remain major scientific challenges. In this respect, network analytical methods have provided a basis for exploring \"hub\" microbial species, which potentially organize community-scale processes of plant-microbe interactions.\n\nMethodsBy compiling Illumina sequencing data of root-associated fungi in eight forest ecosystems across the Japanese Archipelago, we explored hubs within \"metacommunity-scale\" networks of plant-fungus associations. In total, the metadata included 8,080 fungal operational taxonomic units (OTUs) detected from 227 local populations of 150 plant species/taxa.\n\nResultsFew fungal OTUs were common across all the eight forests. However, in each metacommunity-scale network representing northern four localities or southern four localities, diverse mycorrhizal, endophytic, and pathogenic fungi were classified as \"metacommunity hubs\", which were detected from diverse host plant taxa throughout a climatic region. Specifically, Mortierella (Mortierellales), Cladophialophora (Chaetothyriales), Ilyonectria (Hypocreales), Pezicula (Helotiales), and Cadophora (incertae sedis) had broad geographic and host ranges across the northern (cool-temperate) region, while Saitozyma/Cryptococcus (Tremellales/Trichosporonales) and Mortierella as well as some arbuscular mycorrhizal fungi were placed at the central positions of the metacommunity-scale network representing warm-temperate and subtropical forests in southern Japan.\n\nConclusionsThe network theoretical framework presented in this study will help us explore prospective fungi and bacteria, which have high potentials for agricultural application to diverse plant species within each climatic region. As some of those fungal taxa with broad geographic and host ranges have been known to increase the growth and pathogen resistance of host plants, further studies elucidating their functional roles are awaited.

microbiology↗

bioRxiv2018

Dynamics of pellet fragmentation in liquid-grown cultures of Streptomyces lividans

Streptomycetes are extensively used for the production of valuable products, including various antibiotics and industrial enzymes. The preferred way to grow these bacteria in industrial settings is in large-scale fermenters. Growth of streptomycetes under these conditions is characterized by the formation of complex mycelial particles, called pellets. While the process of pellet formation is well characterized, little is known about their disintegration. Here, we use a qualitative and quantitative approach to show that pellet fragmentation in Streptomyces lividans is initiated when cultures enter the stationary phase, which coincides with a remarkable change in pellet architecture. Unlike young pellets, aging pellets have a less dense appearance and are characterized by the appearance of filaments protruding from their outer edges. These morphological changes are accompanied by a dramatic increase in the number of mycelial fragments in the culture broth. In the presence of fresh nutrients, these fragments are able to aggregate with other small fragments, but not with disintegrating pellets, to form new mycelial particles. Altogether, our work indicates that fragmentation might represent an escape mechanism from the environmental stress caused by nutrient scarcity, with striking similarities to the disassembly of bacterial biofilms.

microbiology↗

bioRxiv2018

SAMHD1 Impairs HIV-1 Gene Expression and Reactivation of Viral Latency in CD4+ T-cells

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in non-dividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T-cells that are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 LTR activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T-cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 long terminal repeat (LTR)-driven gene expression at the level of transcription. SAMHD1 overexpression also suppressed LTR activity from human T-cell leukemia virus type 1 (HTLV-1), but not from murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D/R207N, or HD/RN) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro. These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression contributes to regulation of viral latency in CD4+ T-cells.\n\nIMPORTANCEA critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T-cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in non-dividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T-cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T-cells.

microbiology↗

bioRxiv2018

Membrane proteins with high N-glycosylation, high expression, and multiple interaction partners were preferred by mammalian viruses as receptors

Receptor mediated entry is the first step for viral infection. However, the relationship between viruses and receptors is still obscure. Here, by manually curating a high-quality database of 268 pairs of mammalian virus-host receptor interaction, which included 128 unique viral species or sub-species and 119 virus receptors, we found the viral receptors were structurally and functionally diverse, yet they had several common features when compared to other cell membrane proteins: more protein domains, higher level of N-glycosylation, higher ratio of self-interaction and more interaction partners, and higher expression in most tissues of the host. Additionally, the receptors used by the same virus tended to co-evolve. Further correlation analysis between viral receptors and the tissue and host specificity of the virus shows that the virus receptor similarity was a significant predictor for mammalian virus cross-species. This work could deepen our understanding towards the viral receptor selection and help evaluate the risk of viral zoonotic diseases.

microbiology↗

bioRxiv2018

Within-host recombination in structural proteins of the Foot-and-Mouth Disease Virus

Although recombination is known to occur in FMDV, it is considered only a minor determinant of virus sequence diversity. This is because recombination appears to be highly suppressed at phylogenetic scales; inter-serotypic recombination events are rare; and in those a mosaic structure is present whereby recombination only occurs almost exclusively in non-structural proteins. Here we show that co-inoculation of closely related strains in buffaloes results over time in extensive within-host recombination in the genomic region coding for structural proteins. This enables us to directly estimate recombination rates for the first time. Quite surprisingly, the effective recombination rate in VP1 during the acute infection phase turns out to be about 0.1 per base per year, i.e. comparable to the mutation/substitution rate. Thanks to the features of our experimental setup, we are also able to build a high-resolution map of effective within-host recombination in the capsid-coding region. We find that the linkage disequilibrium pattern inside VP1 points to a mosaic structure with two main genetic blocks. Positive epistatic interactions between co-evolved variants appear to be present both within and between blocks. These interactions are due to intra-host selection both at the RNA and protein level. Overall our findings show that during FMDV co-infections by closely related strains, capsid-coding genes recombine within the host at a much higher rate than expected, despite the presence of strong constraints dictated by the capsid structure. Although those intra-host results are not immediately transportable to a phylogenetic setting, they force us to reconsider the relevance of recombination and epistasis, suggesting that they must play a major and so far underappreciated role in the molecular evolution of the virus at all time scales.\n\nAuthor summaryRecombination in the capsid-coding region of the Foot-and-Mouth Disease virus genome is highly suppressed at phylogenetic scales. However, the role of recombination in the intra-host dynamics of the virus is not known. In our experiment, a co-infection of African buffaloes with closely related FMDV strains results in a population structure of the intra-host viral swarm, allowing us to detect recombination events. For structural protein-coding sequences, the swarm dynamics is driven by extensive within-host recombination. During the acute infection phase, we infer intra-host recombination rates of 0.1 per base per year, comparable to the typical mutation rate of the virus. The recombination map reveals two linkage blocks within the VP1 protein-coding sequence. Epistatic interactions between co-evolved mutations in VP1 are caused by intra-host selection at the RNA and protein level and are present both within and between blocks. Our findings support a major role for recombination and epistasis in the intra-host evolution of FMDV.

microbiology↗

bioRxiv2018

Conjugation factors controlling F-plasmid antibiotic resistance transmission

The rapid horizontal transmission of many antibiotic resistance genes between bacterial host cells on conjugative plasmids is a major cause of the accelerating antibiotic resistance crisis. Preventing understanding and targeting conjugation, there currently are no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation. We introduce a novel experimental framework to screen for conjugation based horizontal transmission of antibiotic resistance between >60.000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high throughput. We then mix the resistance plasmid carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explored chromosomal genes controlling F plasmid donation within E. coli populations, by screening the Keio deletion collection at high replication. We recover all six known chromosomal gene mutants affecting conjugation and identify >50 novel factors, all of which diminish antibiotic resistance transmission. We verify 10 of the novel genes effects in a liquid mating assay. The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improves the longevity of current and future antibiotics.

microbiology↗

bioRxiv2018

Multiple Small RNAs Alter Membrane Lipid Composition via Post-Transcriptional Regulation of Cyclopropane Fatty Acid Synthase

Altering membrane protein and lipid composition is an important strategy for maintaining membrane integrity during environmental stress. Many bacterial small RNAs (sRNAs) control membrane protein production, but sRNA-mediated regulation of membrane fatty acid composition is less understood. The sRNA RydC was previously shown to stabilize cfa (cyclopropane fatty acid synthase) mRNA, resulting in higher levels of cyclopropane fatty acids in the cell membrane. Here, we report that three additional sRNAs, ArrS, CpxQ, and GadF also regulate cfa post-transcriptionally. RydC, ArrS, and GadF act through masking an RNase E cleavage site in the cfa mRNA 5 untranslated region (UTR), and all three sRNAs post-transcriptionally activate cfa. In contrast, CpxQ binds to a different site in the cfa mRNA 5 UTR and represses cfa. Alteration of membrane lipid composition is a key mechanism for bacteria to survive low pH environments, and we show that cfa translation increases in an sRNA-dependent manner when cells are subjected to mild acid stress. This work suggests an important role for sRNAs in the acid stress response through regulation of cfa mRNA.

microbiology↗