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bioRxiv2017

A single Vibrionales 16S rRNA oligotype dominates the intestinal microbiome in two geographically separated Atlantic cod populations

BackgroundHost-microbe interactions are particularly intriguing in Atlantic cod (Gadus morhua), as it lacks the MHC II complex involved in presentation of extracellular pathogens. Nonetheless, little is known about the diversity of its microbiome in natural populations. Here, we use 16S rRNA high-throughput sequencing to investigate the microbial community composition in gut content and mucosa of 22 adult individuals from two coastal populations in Norway, located 470 km apart.\n\nResultsWe identify a core microbiome of 23 OTUs (97% sequence similarity) in all individuals that comprises 93% of the total number of reads. The most abundant orders are classified as Vibrionales, Fusobacteriales, Clostridiales and Bacteroidales. While mucosal samples show significantly lower diversity than gut content samples, no differences in OTU community composition are observed between the two populations. The differential abundance of oligotypes within two common OTUs does reveal limited spatial segregation. Remarkably, the most abundant OTU consists of a single oligotype (order Vibrionales, genus Photobacterium) that represents nearly 50% of the reads in both locations.\n\nConclusionsOur results show that the intestinal bacterial community of two geographically separated coastal populations of Atlantic cod is dominated by a limited number of highly abundant 16S rRNA oligotypes shared by all specimens examined. The ubiquity of these oligotypes suggests that the northern coastal Atlantic cod gut microbiome is colonized by a limited number of species with excellent dispersal capabilities that are well suited to thrive in their host environment.

microbiology↗

bioRxiv2017

Genetic and metabolic regulation of Mycobacterium tuberculosis acid growth arrest

Mycobacterium tuberculosis (Mtb) senses and adapts to acidic environments during the course of infection. Acidic pH-dependent adaptations include the induction of metabolic genes associated with anaplerosis and growth arrest on specific carbon sources. In this study, reverse and forward genetic studies were undertaken to define new mechanisms underlying pH-dependent adaptations. Here we report that deletion of isocitrate lyase (icl1/2) or phosphoenolpyruvate carboxykinase (pckA) results in reduced growth at acidic pH and altered metabolite profiles, supporting that remodeling of anaplerotic metabolism is required for pH-dependent adaptation. Mtb cultured at pH 5.7 in minimal medium containing glycerol as a single carbon source exhibits an acid growth arrest phenotype, where the bacterium is non-replicating but viable and metabolically active. The bacterium uptakes and metabolizes glycerol and maintains ATP pools during acid growth arrest and becomes tolerant to detergent stress and the antibiotics isoniazid and rifampin. A forward genetic screen identified mutants that do not arrest their growth at acidic pH, including four enhanced acid growth (eag) mutants with three distinct mutations in the PPE gene MT3221. Overexpression of the MT3221(S211R) variant protein in wild type Mtb results in enhanced acid growth and reduced drug tolerance. Together, these findings provide new evidence for a genetic and physiological basis for acid growth arrest and support that growth arrest is an adaptive process and not simply a physiological limitation associated with acidic pH.\n\nAuthor SummaryThe bacterium Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis in humans. During infection Mtb colonizes a variety of environments that have acidic environments and Mtb must adapt to these environments to cause disease. One of these adaptations is that Mtb slows and arrests its growth at acidic pH, and the goal of this study was to examine the genetics and physiology of these pH-dependent adaptations. We found that Mtb modifies its metabolism at acidic pH and that these adaptations are required for optimal growth. We also found that acidic pH and specific nutrient sources can promote the bacterium to enter a state of dormancy, called acid growth arrest, where the bacterium becomes tolerant to antibiotics. Mutants were identified that do not arrest their growth at acidic, revealing that acid growth arrest is a genetically controlled process. Overall, understanding how Mtb adapts to acidic pH has revealed pathway that are required for virulence and drug tolerance and thus may identify new targets for drug development that may function to shorten the course of TB therapy.

microbiology↗

bioRxiv2017

Regulated release of cryptococcal polysaccharide drives virulence and suppresses immune cell infiltration into the central nervous system

Cryptococcus neoformans is a common environmental yeast and opportunistic pathogen responsible for 15% of AIDS-related deaths worldwide. Mortality primarily results from meningoencephalitis, which occurs when fungal cells disseminate from the initial pulmonary infection site and spread to the brain. A key C. neoformans virulence trait is the polysaccharide capsule. Capsule shields C. neoformans from immune-mediated recognition and destruction. The main capsule component, glucuronoxylomannan (GXM), is found both attached to the cell surface and free in the extracellular space (as exo-GXM). Exo-GXM accumulates in patient serum and cerebrospinal fluid at g/mL concentrations, has well-documented immunosuppressive properties, and correlates with poor patient outcomes. However, it is poorly understood whether exo-GXM release is regulated or the result of shedding during normal capsule turnover. We demonstrate that exo-GXM release is regulated by environmental cues and inversely correlates with surface capsule levels. We identified genes specifically involved in exo-GXM release that do not alter surface capsule thickness. The first mutant, liv7{triangleup}, released less GXM than wild-type cells when capsule is not induced. The second mutant, cnag_00658{triangleup}, released more exo-GXM under capsule-inducing conditions. Exo-GXM release observed in vitro correlated with polystyrene adherence, virulence, and fungal burden during murine infection. Additionally, we find that exo-GXM reduces cell size and capsule thickness in capsule-inducing conditions, potentially influencing dissemination. Finally, we demonstrated that exo-GXM prevents immune cell infiltration into the brain during disseminated infection and highly inflammatory intracranial infection. Our data suggest that exo-GXM performs a different role from capsule GXM during infection, altering cell size and suppressing inflammation.\n\nImportanceCryptococcus neoformans is a leading cause of life-threatening meningoencephalitis in humans. C. neoformans cells produce an immunosuppressive polysaccharide, glucuronoxylomannan (GXM), that is the main component of a protective surface capsule. GXM is also released free into extracellular space as exo-GXM, although the distinction between cell-attached GXM and exo-GXM has been unclear. Exo-GXM influences the outcome of infection, is the basis for current diagnostic tools, and has potential therapeutic applications. This study increases our basic understanding of the fungal biology that regulates polysaccharide release, suggesting that the release of cell-attached GXM and exo-GXM are distinctly regulated. We also introduce a new concept that exo-GXM may alter cell body and capsule size, thereby influencing dissemination in the host. Finally, we provide experimental evidence to confirm clinical observations that exo-GXM influences inflammation during brain infection.

microbiology↗

bioRxiv2017

Viable But Non-Culturable Cells Are Persister Cells

Bacteria have two dormant phenotypes: the viable but non-culturable (VBNC) state and the persister state. Both resting stages arise without mutation and both have been linked to chronic infections; however, persister cells revive rapidly whereas the cell population called VBNC is reported to not resuscitate. Here we investigated the relatedness of the two stress-induced phenotypes at the single-cell level by using transmission electron microscopy and fluorescent microscopy to examine cell morphology and by quantifying cell resuscitation. Using the classic starvation conditions to create VBNC cells, we found that the majority of the remaining Escherichia coli population are spherical, have empty cytosol, and fail to resuscitate; however, some of the spherical cells under these classic VBNC-inducing conditions resuscitate immediately (most probably those with dense cytosol). Critically, all the culturable cells became persister cells within 14 days of starvation. We found that the persister cells initially are rodlike, have clear but limited membrane damage, can resuscitate immediately, and gradually become spherical by aging. After 24 h, only rod-shaped persister cells survive, and all the spherical cells lyse. Both cell populations formed under the VBNC-inducing conditions and the persister cells are metabolically inactive. Therefore, the bacterial population consists of dead cells and persister cells in the VBNC-inducing conditions; i.e., the non-lysed particles that do not resuscitate are dead, and the dormant cells that resuscitate are persister cells. Hence, \"VBNC\" and \"persister\" describe the same dormant phenotype.

microbiology↗

bioRxiv2017

Compensatory evolution facilitates the acquisition of multiple plasmids in bacteria

The coexistence of multicopy plasmids is a common phenomenon. However, the evolutionary forces promoting these genotypes are poorly understood. In this study, we have analyzed multiple ColE1 plasmids (pB1000, pB1005 and pB1006) coexisting within Haemophilus influenzae RdKW20 in all possible combinations. When transformed into the naive host, each plasmid type presented a particular copy number and produced a specific resistance profile and biological cost, whether alone or coexisting with the other plasmids. Therefore, there was no fitness advantage associated with plasmid coexistence that could explain these common plasmid associations in nature. Using experimental evolution, we showed how H. influenzae Rd was able to completely compensate the fitness cost produced by any of these plasmids. Crucially, once the bacterium has compensated for a first plasmid, the acquisition of new multicopy plasmid(s) did not produced any extra biological cost. We argue therefore that compensatory adaptation pave the way for the acquisition of multiple coexisting ColE1 plasmids.\n\nImportanceAntibiotic resistance is a major concern for human and animal health. Plasmids play a major role in the acquisition and dissemination of antimicrobial resistance genes. In this report we investigate, for the first time, how plasmids are capable to cohabit stably in populations. This coexistence of plasmids is driven by compensatory evolution alleviating the cost of a first plasmid, which potentiates the acquisition of further plasmids at no extra cost. This phenomenon explains the high prevalence of plasmids coexistance in wild type bacteria, which generates multiresistant clones and contributes to the maintenance and spread of antibiotic resistance genes within bacterial populations.

microbiology↗

bioRxiv2017

Formation of functional, non-amyloidogenic fibres by recombinant Bacillus subtilis TasA

Bacterial biofilms are communities of microbial cells encased within a self-produced polymeric matrix. In the Bacillus subtilis biofilm matrix the extracellular fibres of TasA are essential. Here a recombinant expression system allows interrogation of TasA, revealing that monomeric and fibre forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibres form spontaneously, and share the biological activity of TasA fibres extracted from B. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophysical properties of both native and recombinant TasA fibres indicate that they are not functional amyloid-like fibres. A gel formed by TasA fibres can recover after physical shear force, suggesting that the biofilm matrix is not static and that these properties may enable B. subtilis to remodel its local environment in response to external cues. Using recombinant fibres formed by TasA orthologues we uncover species variability in the ability of heterologous fibres to cross-complement the B. subtilis tasA deletion. These findings are indicative of specificity in the biophysical requirements of the TasA fibres across different species and/or reflect the precise molecular interactions needed for biofilm matrix assembly.\n\nContributionsConceived and designed the experiments: CE, EE, RG, CEM, RJM, MS, NSW; Performed the experiments: KB, LC, CE, EE, PKF, RG, CEM, RJM, MS, TS; Contributed new analytical tools: CE, EE, RG, TS; Analysed the data: CE, EE, CEM, RJM, MS, LCS, NSW; Wrote the paper: EE, RJM, CEM, MS, NSW.

microbiology↗

bioRxiv2017

N VITRO AND IN VIVO CHARACTERISATION OF ISOLATES OF CRYPTOCOCCUS NEOFORMANS CAUSING MENINGITIS IN HIV-INFECTED AND UNINFECTED PATIENTS IN VIETNAM

We previously observed a substantial burden of cryptococcal meningitis in Vietnam atypically arising in HIV-uninfected individuals. This disease was associated with a single genotype of Cryptococcus neoformans (Sequence Type (ST)5), which was significantly less common in HIV-infected individuals. Aiming to compare the phenotypic characteristics of ST5 and non-ST5 C. neoformans we selected 30 representative Vietnamese isolates, compared their in vitro pathogenic potential and in vivo virulence. ST5 and non-ST5 organisms exhibited comparable characteristics with respect to in vitro virulence markers including melanin production, replication at 37{degrees}C, and growth in cerebrospinal fluid. However, the ST5 isolates had significantly increased variability in cellular and capsular sizing compared with non-ST5 organisms (p<0.001). Counter-intuitively, mice infected with ST5 isolates had significantly longer survival with lower fungal burdens at day 7 than non-ST5 isolates. Notably, ST5 isolates induced significantly greater initial inflammatory responses than non-ST5 strains, measured by TNF- concentrations (p<0.001). Despite being generally less virulent in the mouse model, we hypothesize that the significant within strain variation seen in ST5 isolates in the tested phenotypes may represent an evolutionary advantage enabling adaptation to novel niches including apparently immunocompetent human hosts.

microbiology↗

bioRxiv2017

Pharmacodynamic Considerations of Collateral Sensitivity in Design of Antibiotic Treatment Regimen

Antibiotics have greatly reduced the morbidity and mortality due to infectious diseases. Although antibiotic resistance is not a new problem, its breadth now constitutes a significant threat to human health. One strategy to help combat resistance is to find novel ways to use existing drugs, even those that display high rates of resistance. For the pathogens Escherichia coli and Pseudomonas aeruginosa, pairs of antibiotics have been identified for which evolution of resistance to drug A increases sensitivity to drug B and vice versa. These research groups have proposed cycling such pairs to treat infections, similar treatment strategies are being investigated for various cancer forms as well.\n\nWhile an exciting treatment prospect, no cycling experiments have yet been performed with consideration of pharmacokinetics (PK) and pharmacodynamics (PD). To test the plausibility of this scheme and search for ways to optimize it, we create a mathematical model with explicit PK-PD considerations. We study several possible treatment protocols using pairs of such antibiotics, and investigate the speed of ascent of multiply resistant mutants. Our analyses show that for low concentrations of antibiotics, treatment failure will always occur due to the rapid ascent and fixation of resistant mutants. However, at moderate to high concentrations of some types of bacteriostatic antibiotics with multiday cycling, resistance is prevented from developing and treatment succeeds. This calls for guarded optimism of such treatment protocols whose development can be directed by these types of models.

microbiology↗

bioRxiv2017

Spatial variation of the native colon microbiota in healthy adults

The microbiome has been implicated in the development of colorectal cancer (CRC) and inflammatory bowel diseases (IBD). The specific traits of these diseases vary along the axis of the digestive tract. Further, variation in the structure of the gut microbiota has been associated with both diseases. Here we profiled the microbiota of the healthy proximal and distal mucosa and lumen to better understand how bacterial populations vary along the colon. We used a two-colonoscope approach to sample proximal and distal mucosal and luminal contents from the colons of 20 healthy subjects that had not undergone any bowel preparation procedure. The biopsies and home-collected stool were subjected to 16S rRNA gene sequencing and Random Forest classification models were built using taxa abundance and location to identify microbiota specific to each site. The right mucosa and lumen had the most similar community structures of the five sites we considered from each subject. The distal mucosa had higher relative abundance of Finegoldia, Murdochiella, Peptoniphilus, Porphyromonas and Anaerococcus. The proximal mucosa had more of the genera Enterobacteriaceae, Bacteroides and Pseudomonas. The classification model performed well when classifying mucosal samples into proximal or distal sides (AUC = 0.808). Separating proximal and distal luminal samples proved more challenging (AUC = 0.599) and specific microbiota that differentiated the two were hard to identify. By sampling the unprepped colon, we identified distinct bacterial populations native to the proximal and distal sides. Further investigation of these bacteria may elucidate if and how these groups contribute to different disease processes on their respective sides of the colon.

microbiology↗

bioRxiv2017

Isolation and identification of enteroviruses from sewage and sewage-contaminated water samples from Ibadan, Nigeria, 2012-2013.

In 2010, we described sewage contaminated water (SCW) bodies that consistently yielded enteroviruses (EVs) in enterovirus surveillance (ES) sites in Lagos, Nigeria. By 2012, we demonstrated the presence and circulation of Wild Poliovirus 3 (WPV3) in these ES sites. Here we describe ES sites that consistently yield EVs in Ibadan metropolis southwest Nigeria.\n\nTwenty-five ES samples were collected by grab method from nine sites between October, 2012 and March, 2013. Samples were concentrated and four (RD, HEp2C, MCF-7 and L20B) different cell lines used for virus isolation from the concentrates. Isolates were subjected to RNA extraction, cDNA synthesis, PanEnterovirus 5l-UTR and VP1 assays. Unidentifiable isolates were further subjected to species-specific RT-PCR assays. Amplicons were sequenced, isolates identified and subjected to phylogenetic analysis.\n\nTwenty-five isolates were recovered from 8 (32%) of the 25 ES samples collected. Twenty-three of the isolates were identified as EVs by the PanEntero5l-UTR assay. Thirteen (57%) of the 23 EVs were positive for the VP1 assay, and identified as Coxsackievirus B3 (CVB3) (1 isolate), CVB6 (1 isolate), E6 (2 isolates), E7 (5 isolates), E11 (1 isolate), E12 (1 isolate) and E13 (2 isolates). None and 2 (25%) of the remaining isolates were positive for the EV-B and EV-C assays, respectively. The 2 EV-C positive enteroviruses were isolated on MCF-7.\n\nThis study describes three very productive ES sites, and documents the presence of CVB3, CVB6, E6, E7, E11, E12 and, E13 in Ibadan, Nigeria. It shows that including other cell lines in EV isolation protocols can broaden the diversity of EV types recoverable.

microbiology↗

bioRxiv2017

The Gram-positive model organism Bacillus subtilis does not form detectable cardiolipin-specific lipid domains

Rather than being a homogenous diffusion-dominated structure, biological membranes can exhibit areas with distinct composition and characteristics commonly termed as lipid domains. Arguably the most comprehensively studied examples in bacteria are domains formed by cardiolipin, which have been functionally linked to protein targeting, cell division process, and mode of action of membrane targeting antimicrobials. Cardiolipin domains were originally identified in the Gram-negative model organism Escherichia coli based on preferential staining by the fluorescent membrane dye nonyl acridine orange (NAO), and later reported to exist also in other Gram-negative and -positive bacteria. Recently, the lipid-specificity of NAO has been questioned based on studies conducted in E. coli. This prompted us to re-analyse cardiolipin domains also in the Gram-positive model organism Bacillus subtilis. Here we show that logarithmically growing B. subtilis does not form microscopically detectable cardiolipin-specific lipid domains, and that NAO is not a specific stain for cardiolipin in this organism.\n\nAbbreviations

microbiology↗

bioRxiv2017

The differentially expressed translationally controlled tumor proteins TCTP1 and TCTP2 in Trypanosoma brucei

1. SummaryIn Trypanosoma brucei we identified two TCTP genes differentially expressed during the parasite life cycle and discovered the mechanism by which this expression is controlled. Furthermore, we demonstrate that TCTP is important for cell growth as well as proper cell and organelle morphology in the insect form of the parasite.\n\n2. AbstractTCTP is a highly conserved protein ubiquitously expressed in eukaryotes. Studies have reported TCTP to be involved in growth and development, cell cycle progression, protection against cellular stresses and apoptosis, indicating the multifunctional role of the protein. Here, for the first time we characterize the expression and function of TCTP in the unicellular parasite Trypanosoma brucei. We identified two paralogue TCTP genes, which we named TbTCTP1 and TbTCTP2. They have identical 5UTRs and only ten single nucleotide polymorphisms in the open reading frames (ORFs). However, the 3UTRs differ dramatically in sequence and length. We found that the two TCTP mRNAs are differentially expressed during T. brucei life cycle. While procyclic form trypanosomes (PCF) express TCTP1, the bloodstream form trypanosomes (BSF), express TCTP2. We link the differential expression to the distinct 3UTRs of the paralogues. In PCF cells, the protein appears to localize in the cytosol. We show that TCTP1 is essential for normal cell growth and has pleiotropic effects on the cells including aberrant cell morphology, enlarged and reduced number of acidocalcisomes and appearance of accumulations in the mitochondria.

microbiology↗

bioRxiv2017

The virome of Drosophila suzukii, an invasive pest of soft fruit

Drosophila suzukii (Matsumura) is one of the most damaging and costly pests to invade temperate horticultural regions in recent history. Conventional control of this pest is challenging, and an environmentally benign microbial biopesticide is highly desirable. A thorough exploration of the pathogens infecting this pest is not only the first step on the road to the development of an effective biopesticide, but also provides a valuable comparative dataset for the study of viruses in the model family Drosophilidae. Here we use a metatransciptomic approach to identify viruses infecting this fly in both its native (Japanese) and invasive (British and French) ranges. We describe 18 new RNA viruses, including members of the Picornavirales, Mononegavirales, Bunyavirales, Chuviruses, Nodaviridae, Tombusviridae, Reoviridae, and Nidovirales, and discuss their phylogenetic relationships with previously known viruses. We also detect 18 previously described viruses of other Drosophila species that appear to be associated with D. suzukii in the wild.

microbiology↗

bioRxiv2017

Methods matter: Influential purification and analysis parameters for intracellular parasite metabolomics

Metabolomics is increasingly popular for the study of many pathogens. For the malaria parasite, Plasmodium falciparum, both targeted and untargeted metabolite detection has improved our understanding of pathogenesis, host-parasite interactions, and antimalarial drug treatment and resistance. However, purification and analysis procedures for performing metabolomics on intracellular pathogens have not been explored. Here, we investigate the impact of host contamination on the metabolome when preparing samples using standard methods. We purified in vitro grown ring stage intra-erythrocytic P. falciparum parasites for untargeted metabolomics studies; the small size of this developmental stage amplifies the challenges associated with metabolomics studies as the ratio between host and parasite biomass is maximized. Following metabolite identification and data preprocessing, we investigated whether host contributions could be corrected post hoc using various normalization approaches (including double stranded DNA, total protein, or parasite number). We conclude that normalization parameters have large effects on differential abundance analysis and recommend the thoughtful selection of these parameters. However, normalization does not remove the contribution from the parasites extracellular environment (culture media and host erythrocyte). In fact, we found that extra-parasite material is as influential on the metabolome as treatment with a potent antimalarial drug with known metabolic effects (artemisinin). Because of this influence, we could not detect significant changes associated with drug treatment. Instead, we identified metabolites predictive of host and media contamination that can be used to assess sample purification. Our findings provide a basis for development of improved experimental and analytical methods for future metabolomics studies of intracellular organisms.

microbiology↗

bioRxiv2017

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis

Fungi undergo changes in cell shape in response to environmental stimuli that drive pathogenesis and niche adaptation, such as the yeast-to-hyphal transition of dimorphic fungi in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify bacterial peptidoglycan as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and E. coli co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established and previously undescribed regulators of Titanisation in vitro and investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-titan transition and establish an essential in vitro model for the future characterization of this important morphotype.\n\nAuthor SummaryChanges in cell shape underlie fungal pathogenesis by allowing immune evasion and dissemination. Aspergillus and Candida albicans hyphae drive tissue penetration. Histoplasma capsulatum and C. albicans yeast growth allows evasion and dissemination. As major virulence determinates, morphogenic transitions are extensively studied in animal models and in vitro. The pathogenic fungus Cryptococcus neoformans is a budding yeast that, in the host lung, switches to an unusual morphotype termed the Titan cell. Titans are large, polyploid, have altered cell wall and capsule, and produce haploid daughters. Their size prevents engulfment by phagocytes, yet they are linked to dissemination and altered immune response. Despite their important influence on disease, replicating the yeast-to-Titan switch in vitro has proved challenging. Here we show that Titans are induced by host-relevant stimuli, including serum and bronchio-alveolar lavage fluid. We identify bacterial peptidoglycan as a relevant inducing compound and predict an in vivo Titan defect for a clinical isolate. Genes regulating in vivo Titanisation also influence in vitro formation. Titanisation is a conserved morphogenic switch across the C. neoformans species complex. Together, we show that Titan cells are a regulated morphotype analogous to the yeast-to-hyphal transition and establish new ways to study Titans outside the host lung.

microbiology↗

bioRxiv2017

Loss of bone marrow cells including CD34+ cells following CCR5-tropic HIV-1 infection of humanized NOD/SCID/JAK3null mice and partial protection of those cells by a promotor-targeting shRNA

ObjectivesHematological abnormalities that include changes in bone marrow, such as in anemia and pancytopenia, are common among human immunodeficiency virus (HIV)-infected patients, particularly in the advanced stage of disease. This study aimed to provide better experimental evidence of such manifestations in animal models.\n\nDesignNOD/SCID/JAK3null (NOJ) mice were transplanted with human cord-derived CD34+ cells with or without transduction with a lentiviral vector expressing a promoter-targeting shRNA called PromA.\n\nMethodsAt 16 weeks after transplantation, mice engrafted with CD34+ cells were infected with CCR5-tropic HIV-1JRFL.\n\nResultsAt week 2 post infection, HIV replication was observed in peripheral blood mononuclear cells and splenocytes. In mice transplanted with unmanipulated CD34+ cells, viral replication was accompanied by a loss of peripheral/spleen CD4+CCR5+ T cells. Interestingly, bone marrow CD34+ cells in HIV-infected mice were also depleted, but in a CXCR4-associated manner. Conversely, the lentiviral transfer of PromA in CD34+ cells prior to transplantation rendered the humanized NOJ mice resistant to HIV replication in CD4+ T cells, resulting in better preservation of peripheral/spleen CD4+CCR5+ T cells and bone marrow CD34+ cells at two weeks after infection.\n\nConclusionsThese results implicate the importance of evaluating hematopoietic stem/progenitor cell pools in addition to peripheral CD4+ T-cell counts to assess the early stage of HIV infection. Moreover, stable gene transfer of PromA to hematopoietic stem cells not only limited HIV replication but also led to preservation of different subsets of hematopoietic cells, including bone marrow stem/progenitor cells.

microbiology↗

bioRxiv2017

Cryo-EM reconstruction of AlfA from Bacillus subtilis reveals the structure of a simplified actin-like filament at 3.4 A resolution

Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB and the centromeric sequence parN. Similar to the ParMRC partitioning system from E. coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids, through interactions with AlfB and parN. Since, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of non-bundling AlfA filaments to 3.4 [A] resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin-fold has been accommodated by unique longitudinal and lateral contacts, whilst still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments we obtained a pseudo-atomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly anti-phasic with 8-fold integer helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, signs of the strong evolutionary pressure for simplicity, placed on plasmids: deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerisation, nucleotide hydrolysis and antiparallel doublet formation are retained to fulfil the system's biological raison d'etre.\n\nSignificance StatementProtein filaments perform a vast array of functions inside almost all living cells. Actin-like proteins in archaea and bacteria have previously been found to form a surprising diversity of filament architectures, reflecting their divergent cellular roles. Actin-like AlfA is unique in that it is much smaller than all other filament forming actin-like proteins. With an atomic structure of the AlfA filament, obtained by high-resolution electron cryo-microscopy, we have revealed--at atomic level of detail--how AlfA filaments form dynamic filaments capable of transporting plasmid DNA in cells and how these filaments arrange into antiparallel bundles required for the segregation mechanism.

microbiology↗

bioRxiv2017

Decoupling of the nuclear division cycle and cell size control in the coenocytic cycle of the ichthyosporean Sphaeroforma arctica

Coenocytes (multinucleated cells formed by sequential nuclear divisions without cytokinesis) are commonly found across the eukaryotic kingdom, including in animals, plants and several lineages of unicellular eukaryotes. Despite their commonality, little is known about how cell growth, nuclear divisions and cell divisions are coordinated in coenocytes. Among the unicellular eukaryotes that form coenocytes are ichthyosporeans, a lineage of unicellular holozoans that are of significant interest due to their phylogenetic placement as one of the closest relatives to animals. Here, we characterize the coenocytic cell division cycle in the ichthyosporean Sphaeroforma arctica. In laboratory conditions, we observed that S. arctica cells undergo a highly regular periodic coenocytic cell cycle. Nuclear division cycles occur synchronously within the coenocyte and in regular time intervals (~11 hours per nuclear cycle) until reaching 64-128 nuclei and releasing daughter cells. The duration of the nuclear division cycles is constant across a wide range of nutrient concentration. In contrast, the volume of the coenocytes increase more slowly in lower nutrient concentration, which also results in smaller newborn daughter cells. This suggests that S. arctica cells are capable to adapt the cell growth rate to nutrient concentration while maintaining the timing of nuclear division cycles, suggesting that in ichthyosporeans the mechanisms regulating highly periodic nuclear division cycles operate independently from mechanisms sensing the cell size.

microbiology↗