Wild-type Parent Strain Research Articles

Mechanosensitive channel MscS is critical for termination of the bacterial hypoosmotic permeability response.

Free-living microorganisms are subjected to drastic changes in osmolarity. To avoid lysis under sudden osmotic down-shock, bacteria quickly expel small metabolites through the tension-activated channels MscL, MscS, and MscK. We examined five chromosomal knockout strains, ∆mscL, ∆mscS, a double knockout ∆mscS ∆mscK, and a triple knockout ∆mscL ∆mscS ∆mscK, in comparison to the wild-type parental strain. Stopped-flow experiments confirmed that both MscS and MscL mediate fast osmolyte release and curb cell swelling, but osmotic viability assays indicated that they are not equivalent. MscS alone was capable of rescuing the cell population, but in some strains, MscL did not rescue and additionally became toxic in the absence of both MscS and MscK. Furthermore, MscS was upregulated in the ∆mscL strain, suggesting either a crosstalk between the two genes/proteins or the influence of cell mechanics on mscS expression. The data shows that for the proper termination of the permeability response, the high-threshold (MscL) and the low-threshold (MscS/MscK) channels must act sequentially. In the absence of low-threshold channels, at the end of the release phase, MscL should stabilize membrane tension at around 10 mN/m. Patch-clamp protocols emulating the tension changes during the release phase indicated that the non-inactivating MscL, residing at its own tension threshold, flickers and produces a protracted leakage. The MscS/MscK population, when present, stays open at this stage to reduce tension below the MscL threshold and silence the large channel. When MscS reaches its own threshold, it inactivates and thus ensures proper termination of the hypoosmotic permeability response. This functional interplay between the high- and low-threshold channels is further supported by the compromised osmotic survival of bacteria expressing non-inactivating MscS mutants.

Role of the Extracytoplasmic Function Sigma Factor SigE in the Stringent Response of Mycobacterium tuberculosis.

Bacteria respond to nutrient starvation implementing the stringent response, a stress signaling system resulting in metabolic remodeling leading to decreased growth rate and energy requirements. A well-characterized model of stringent response in Mycobacterium tuberculosis is the one induced by growth in low phosphate. The extracytoplasmic function (ECF) sigma factor SigE was previously suggested as having a key role in the activation of stringent response. In this study, we challenge this hypothesis by analyzing the temporal dynamics of the transcriptional response of a sigE mutant and its wild-type parental strain to low phosphate using RNA sequencing. We found that both strains responded to low phosphate with a typical stringent response trait, including the downregulation of genes encoding ribosomal proteins and RNA polymerase. We also observed transcriptional changes that support the occurring of an energetics imbalance, compensated by a reduced activity of the electron transport chain, decreased export of protons, and a remodeling of central metabolism. The most striking difference between the two strains was the induction in the sigE mutant of several stress-related genes, in particular, the genes encoding the ECF sigma factor SigH and the transcriptional regulator WhiB6. Since both proteins respond to redox unbalances, their induction suggests that the sigE mutant is not able to maintain redox homeostasis in response to the energetics imbalance induced by low phosphate. In conclusion, our data suggest that SigE is not directly involved in initiating stringent response but in protecting the cell from stress consequent to the low phosphate exposure and activation of stringent response. IMPORTANCE Mycobacterium tuberculosis can enter a dormant state enabling it to establish latent infections and to become tolerant to antibacterial drugs. Dormant bacteria's physiology and the mechanism(s) used by bacteria to enter dormancy during infection are still unknown due to the lack of reliable animal models. However, several in vitro models, mimicking conditions encountered during infection, can reproduce different aspects of dormancy (growth arrest, metabolic slowdown, drug tolerance). The stringent response, a stress response program enabling bacteria to cope with nutrient starvation, is one of them. In this study, we provide evidence suggesting that the sigma factor SigE is not directly involved in the activation of stringent response as previously hypothesized, but it is important to help the bacteria to handle the metabolic stress related to the adaptation to low phosphate and activation of stringent response, thus giving an important contribution to our understanding of the mechanism behind stringent response development.

Tetracarboxylic acid transporter regulates growth, conidiation, and carbon utilization in Metarhizium acridum.

Carbon sources and their utilization are vital for fungal growth and development. C4-dicarboxylic acids are important carbon and energy sources that function as intermediate products of the tricarboxylic acid cycle. Transport and regulation of C4-dicarboxylic acid uptake are mainly dependent on tetracarboxylic acid transporters (Dcts) in many microbes, although the roles of Dct genes in fungi have only been partially characterized. Here, we report on the functions of two Dct genes (Dct1 and Dct2) in the entomopathogenic fungus Metarhizium acridum. Our data showed that loss of the MaDct1 gene affected utilization of tetracarboxylic acids and other carbon sources. ΔMaDct1 mutants showed larger colony sizes with extensive mycelial growth but were delayed in conidiation with decreased conidia yield as compared to the wild-type parental strain. On the nutrient-deficient medium, SYA, the wild-type strain produced microcycle conidia, whereas the ΔMaDct1 mutant produced (normal) aerial conidia. In addition, ΔMaDct1 had decreased tolerance to cell wall perturbing agents, but increased tolerances to UV-B radiation and osmotic stress. Insect bioassays indicated that loss of MaDct1 did not affect pathogenicity. In contrast, no distinct phenotypic change was observed for the MaDct2 mutant in terms of growth and biocontrol characteristics. Transcriptomic profiling between wild type and ΔMaDct1 showed that differentially expressed genes were enriched in carbohydrate and amino acid metabolism, transport and catabolism, and signal transduction. These results demonstrate that MaDct1 regulates the conidiation pattern shift and mycelial growth by affecting utilization of carbon sources. These findings are helpful for better understanding the effect of intermediates of carbon metabolism on fungal growth and conidiation. KEY POINTS: • MaDct1 influences fungal growth and conidiation by affecting carbon source utilization. • MaDct1 regulates conidiation pattern shift under nutrient deficiency condition. • MaDct1 is involved in stress tolerance and has no effect on virulence. • MaDct2 has no effect on growth and biocontrol characteristic.

Cdc14 phosphatase contributes to cell wall integrity and pathogenesis in Candida albicans.

The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. Here, we identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm ) as the sole source of Cdc14 proliferated like the wild-type parent strain but exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a novel and conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reduced Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.

Live Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Undergoes an Abortive Replication Cycle in the TG Neurons following Latency Reactivation.

Like other alpha herpesviruses, pseudorabies virus (PRV) establishes lifelong latency in trigeminal ganglionic (TG) neurons. Upon stress, the latent viruses in the TG neurons reactivate and are transported anterograde from the neuron cell bodies to the nerve endings in the nasal mucosa, where they replicate and are discharged in the nasal and oral secretions. Consequently, the virus is transmitted to other naïve animals. This cycle of latency and reactivation continues until the animal dies or is slaughtered. We have constructed a PRV triple mutant virus (PRVtmv) and used it as a live subunit vaccine vector against porcine circovirus 2b (PCV2b) and classical swine fever virus (CSFV) (PRVtmv+). We compared the latency reactivation properties of PRVtmv+ with its parent wild-type (wt) Becker strain following intranasal infection. The results showed that PRV wt and PRVtmv+ established latency in the TG neurons. Based on nasal virus shedding, immediate early (infected cell protein 0; ICP0) and late genes, MCP (major capsid protein) and gC (glycoprotein C) transcriptions, and viral DNA copy numbers in the TGs of latently infected and dexamethasone (Dex)-treated pigs, both PRV wt and PRVtmv+ reactivated from latency. We noticed that PRV wt virus replicated productively in the terminally differentiated, postmitotic TG neurons, but PRVtmv+ failed to replicate and, therefore, there was no virus production in the TG. In addition, we found that only the PRV wt virus was shed in the nasal secretions following the Dex-induced reactivation. Our results demonstrated that the PRVtmv+ is safe as a live viral subunit vaccine vector without the possibility of productive replication in the TG upon reactivation from latency and without subsequent nasal virus shedding. This property of PRVtmv+ precludes the possibility of vaccine virus circulation in pigs and the risk of reversion to virulence.

Triazine-Based Small Molecules: A Potential New Class of Compounds in the Antifungal Toolbox.

Invasive fungal infections caused by Candida species remain a significant public health problem worldwide. The increasing prevalence of drug-resistant infections and a limited arsenal of antifungal drugs underscore the need for novel interventions. Here, we screened several classes of pharmacologically active compounds against mammalian diseases for antifungal activity. We found that the synthetic triazine-based compound melanogenin (Mel) 56 is fungicidal in Candida albicans laboratory and clinical strains with minimal inhibitory concentrations of 8−16 µg/mL. Furthermore, Mel56 has general antifungal activity in several non-albicans Candida species and the non-pathogenic yeast Saccharomyces cerevisiae. Surprisingly, Mel56 inhibited the yeast-to-hyphae transition at sublethal concentrations, revealing a new role for triazine-based compounds in fungi. In human cancer cell lines, Mel56 targets the inner mitochondrial integral membrane prohibitin proteins, PHB1 and PHB2. However, Mel56 treatment did not impact C. albicans mitochondrial activity, and antifungal activity was similar in prohibitin single, double, and triple homozygous mutant strains compared to the wild-type parental strain. These results suggests that Mel56 has a novel mechanism-of-action in C. albicans. Therefore, Mel56 is a promising antifungal candidate warranting further analyses.

Affinity of cefotiam for the alternative penicillin binding protein PBP3SAL used by Salmonella inside host eukaryotic cells.

Following the invasion of eukaryotic cells, Salmonella enterica serovar Typhimurium replaces PBP2/PBP3, main targets of β-lactam antibiotics, with PBP2SAL/PBP3SAL, two homologue peptidoglycan synthases absent in Escherichia coli. PBP3SAL promotes pathogen cell division in acidic environments independently of PBP3 and shows low affinity for β-lactams that bind to PBP3 such as aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefalotin. To find compounds with high affinity for PBP3SAL to control Salmonella intracellular infections. An S. Typhimurium ΔPBP3 mutant that divides using PBP3SAL and its parental wild-type strain, were exposed to a library of 1520 approved drugs in acidified (pH 4.6) nutrient-rich LB medium. Changes in optical density associated with cell filamentation, a read-out of blockage in cell division, were monitored. Compounds causing filamentation in the ΔPBP3 mutant but not in wild-type strain-the latter strain expressing both PBP3 and PBP3SAL in LB pH 4.6-were selected for further study. The bactericidal effect due to PBP3SAL inhibition was evaluated in vitro using a bacterial infection model of cultured fibroblasts. The cephalosporin cefotiam exhibited higher affinity for PBP3SAL than for PBP3 in bacteria growing in acidified LB pH 4.6 medium. Cefotiam also proved to be effective against intracellular Salmonella in a PBP3SAL-dependent manner. Conversely, cefuroxime, which has higher affinity for PBP3, showed decreased effectiveness in killing intracellular Salmonella. Antibiotics with affinity for PBP3SAL, like the cephalosporin cefotiam, have therapeutic value for treating Salmonella intracellular infections.

Mutations in troABCD against Copper Overload in a copA Mutant of Streptococcus suis.

Streptococcus suis is a major swine pathogen that is increasingly recognized as a porcine zoonotic pathogen that threatens the health of both pigs and humans. Metal homeostasis plays a critical role during the process of bacterial infection. In this study, RNA sequencing was used to identify potential candidate genes involved in the maintenance of intracellular copper homeostasis. CopA was identified as the primary copper exporter in S. suis. The copA deletion mutant strain was found to be more sensitive to copper and accumulated more intracellular copper than the wild-type (WT) parent strain. In addition, adding manganese increased the ability of S. suis to resist copper, and the manganese transporter, TroABCD, was involved in tolerance to copper. The copA deletion mutant strain accumulated less copper when supplemented with manganese. Furthermore, when cultured with copper, the double deletion mutant (ΔcopAΔtroA) exhibited improved growth compared to the copA deletion mutant strain. In addition, the double deletion mutant (ΔcopAΔtroA) accumulated less copper than the copA deletion mutant strain. These data were consistent with a model wherein defective TroABCD resulted in decreased cellular copper accumulation and protected the strain against copper poisoning. IMPORTANCE Metal homeostasis plays a critical role during the process of bacterial infection. We identified three important potential candidate genes involved in maintenance of intracellular copper homeostasis. CopA was demonstrated to be the main copper exporter in Streptococcus suis, and manganese increased the tolerance of S. suis to copper. The double deletion mutant (ΔcopAΔtroA) improved growth ability over the copA deletion mutant strain in the presence of high concentrations of copper and accumulated less copper. These findings are consistent with a model wherein defective TroABCD resulted in decreased cellular accumulation of copper and protected the strain against copper poisoning.

Rhizopine biosensors for plant-dependent control of bacterial gene expression.

Engineering signalling between plants and microbes could be exploited to establish host-specificity between plant-growth-promoting bacteria and target crops in the environment. We previously engineered rhizopine-signalling circuitry facilitating exclusive signalling between rhizopine-producing (RhiP) plants and model bacterial strains. Here, we conduct an in-depth analysis of rhizopine-inducible expression in bacteria. We characterize two rhizopine-inducible promoters and explore the bacterial host-range of rhizopine biosensor plasmids. By tuning the expression of rhizopine uptake genes, we also construct a new biosensor plasmid pSIR05 that has minimal impact on host cell growth in vitro and exhibits markedly improved stability of expression in situ on RhiP barley roots compared to the previously described biosensor plasmid pSIR02. We demonstrate that a sub-population of Azorhizobium caulinodans cells carrying pSIR05 can sense rhizopine and activate gene expression when colonizing RhiP barley roots. However, these bacteria were mildly defective for colonization of RhiP barley roots compared to the wild-type parent strain. This work provides advancement towards establishing more robust plant-dependent control of bacterial gene expression and highlights the key challenges remaining to achieve this goal.

Gain-of-function mutant of movement protein allows systemic transport of a defective tobacco mosaic virus.

Functional compensation in response to gene dysfunction is a fascinating phenomenon that allows mutated viruses to regain the capabilities of their wild-type parental strains. In this study, we isolated mutants of tobacco mosaic virus capable of CP-independent systemic movement. These gain-of-function mutants lacked the 16 C-terminal amino acids of the movement protein (MP). Whereas this deletion did not affect the cell-to-cell movement of MP, it dramatically enhanced the viral genomic RNA levels and MP accumulation within the infected cells and altered the subcellular localization of MP from exclusively plasmodesmata (PD) to both PD and plasma membrane. The adapted defective virus suppressed the expression of the ethylene pathway and phloem-associated resistance factors in the inoculated leaves. These findings demonstrate the potential for plant viral MPs to gain a new function that allows viral genomes to move systemically in the absence of the natural viral factor that mediates this spread.

Virulence factors of Mycoplasma synoviae: Three genes influencing colonization, immunogenicity, and transmissibility.

Infections caused by Mycoplasma synoviae are major welfare and economic concerns in poultry industries worldwide. These infections cause chronic respiratory disease and/or synovitis in chickens and turkeys leading to reduced production and increased mortality rates. The live attenuated vaccine strain MS-H (Vaxsafe® MS), commonly used for protection against M. synoviae infection in many countries, contains 32 single nucleotide variations compared to its wildtype parent strain, 86079/7NS. Genomic analysis of vaccine strains reisolated from flocks following the administration of MS-H has identified reversions to the original 86079/7NS sequence in the obgE, oppF and gapdh genes. Here, three MS-H field reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), as well as the vaccine MS-H and the parental strain 86079/7NS were experimentally inoculated to chickens. The strains were assessed for their ability to infect and elicit immune responses in the recipient chickens, as well as in naïve in-contact chickens. Despite the loss of temperature sensitivity phenotype and colonization of the reisolates in the lower respiratory tract, there was no significant differences detected in the microscopic mucosal thickness of the middle or lower trachea of the inoculated chickens. Concurrent reversions in ObgE, OppF and GAPDH proteins were associated with higher gross air sac lesion scores and increased microscopic upper-tracheal mucosal thickness in chickens directly inoculated with the reisolates following intratracheal administration of a virulent strain of infectious bronchitis virus. The gross air sac lesions of the chickens in-contact with those inoculated with reisolates were not significantly different to those of chickens in-contact with MS-H inoculated chickens, suggesting that horizontal transmission of the reisolates in the poultry flock will not lead to higher pathogenicity or clinical signs. These results suggest a significant role of GAPDH and/or cumulative effect of ObgE, OppF and GAPDH on M. synoviae pathogenicity. Future experiments will be required to investigate the effect of single mutations in gapdh or oppF gene on pathogenicity of M. synoviae.

Outer membrane protein of OmpF contributes to swimming motility, biofilm formation, osmotic response as well as the transcription of maltose metabolic genes in Citrobacter werkmanii.

Bacterial outer membrane proteins (Omps) are essential for environmental sensing, stress responses, and substance transport. Our previous study discovered that OmpA contributes to planktonic growth, biocide resistance, biofilm formation, and swimming motility in Citrobacter werkmanii, whereas the molecular functions of OmpF in this strain are largely unknown. Thus, in this study, the ompF gene was firstly knocked out from the genome of C. werkmanii using a homologous recombination method, and its phenotypical alternations of ∆ompF were then thoroughly characterized using biochemical and molecular approaches with the parental wild type (WT) and complementary (∆ompF-com) strains. The results demonstrated that the swimming ability of ∆ompF on semi-solid plates was reduced compared to WT due to the down-regulation of flgC, flgH, fliK, and fliF. Meanwhile, ompF deletion reduces biofilm formation on both glass and polystyrene surfaces due to decreased cell aggregation. Furthermore, ompF inactivation induced different osmotic stress (carbon sources and metal ions) responses in its biofilms when compared to WT and ∆ompF-com. Finally, a total of 6 maltose metabolic genes of lamB, malE, malK, malG, malM, and malF were all up-regulated in ∆ompF. The gene knockout and HPLC results revealed that the MalEFGK2 cluster was primarily responsible for maltose transport in C. werkmanii. Furthermore, we discovered for the first time that the upstream promoter of OmpF and its transcription can be combined with and negatively regulated by MalT. Overall, OmpF plays a role in a variety of biochemical processes and molecular functions in C. werkmanii, and it may even act as a targeted site to inhibit biofilm formation.

Multiple immunodominant O-epitopes co-expression in live attenuated Salmonella serovars induce cross-protective immune responses against S. Paratyphi A, S. Typhimurium and S. Enteritidis.

Salmonella enterica subsp. enterica (S. enterica) is a significant public health concern and is estimated to cause more than 300,000 deaths annually. Nowadays, the vaccines available for human Salmonellosis prevention are all targeting just one serovar, i.e., S. Typhi, leaving a huge potential risk of Salmonella disease epidemiology change. In this study, we explored the strategy of multiple immunodominant O-epitopes co-expression in S. enterica serovars and evaluated their immunogenicity to induce cross-immune responses and cross-protections against S. Paratyphi A, S. Typhimurium and S. Enteritidis. We found that nucleotide sugar precursors CDP-Abe and CDP-Par (or CDP-Tyv) could be utilized by S. enterica serovars simultaneously, exhibiting O2&O4 (or O4&O9) double immunodominant O-serotypes without obvious growth defects. More importantly, a triple immunodominant O2&O4&O9 O-serotypes could be achieved in S. Typhimurium by improving the substrate pool of CDP-Par, glycosyltransferase WbaV and flippase Wzx via a dual-plasmid overexpressing system. Through immunization in a murine model, we found that double or triple O-serotypes live attenuated vaccine candidates could induce significantly higher heterologous serovar-specific antibodies than their wild-type parent strain. Meanwhile, the bacterial agglutination, serum bactericidal assays and protection efficacy experiments had all shown that these elicited serum antibodies are cross-reactive and cross-protective. Our work highlights the potential of developing a new type of live attenuated Salmonella vaccines against S. Paratyphi A, S. Typhimurium and S. Enteritidis simultaneously.

Safety and immunogenicity of four-segmented Rift Valley fever virus in the common marmoset

Rift Valley fever virus (RVFV) is an emerging mosquito-borne bunyavirus that is highly pathogenic to wild and domesticated ruminants, camelids, and humans. While animals are exclusively infected via mosquito bites, humans can also be infected via contact with contaminated tissues or blood. No human vaccine is available and commercialized veterinary vaccines do not optimally combine efficacy with safety. We previously reported the development of two novel live-attenuated RVF vaccines, created by splitting the M genome segment and deleting the major virulence determinant NSs. The vaccine candidates, referred to as the veterinary vaccine vRVFV-4s and the human vaccine hRVFV-4s, were shown to induce protective immunity in multiple species after a single vaccination. Anticipating accidental exposure of humans to the veterinary vaccine and the application of hRVFV-4s to humans, the safety of each vaccine was evaluated in the most susceptible nonhuman primate model, the common marmoset (Callithrix jacchus). Marmosets were inoculated with high doses of each vaccine and were monitored for clinical signs as well as for vaccine virus dissemination, shedding, and spreading to the environment. To accurately assess the attenuation of both vaccine viruses, separate groups of marmosets were inoculated with the parent wild-type RVFV strains. Both wild-type strains induced high viremia and disseminated to primary target organs, associated with mild-to-severe morbidity. In contrast, both vaccines were well tolerated with no evidence of dissemination and shedding while inducing potent neutralizing antibody responses. The results of the studies support the unprecedented safety profile of both vaccines for animals and humans.

The UPC2 gene in Kluyveromyces lactis stress adaptation.

KlUpc2p, atranscription factor belonging to the fungal binuclear cluster family, is an important regulator of ergosterol biosynthesis and azole drug resistance inKluyveromyces lactis.In this work, we show that the absence ofKlUpc2p generates Rag-phenotype and modulates theK. lactissusceptibility to oxidants and calcofuor white. TheKlUPC2deletion leads to increased expression of KlMGA2gene,encoding an important regulator of hypoxic and lipid biosynthetic genes inK. lactis and also KlHOG1 gene. The absence ofKlUpc2p does not lead to statistically significant changes in glycerol, corroborating the expression ofKlGPD1gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase, that is similar in both the deletion mutant and the parental wild-type strain. Increased sensitivity ofKlupc2mutant cells to brefeldin Aaccompanied with significant increase inKlARF2gene expression point to the involvement ofKlUpc2p in intracellular signaling. Our observations highlight the connections between ergosterol and fatty acid metabolism to modulate membrane properties and point to the possible involvement of KlUpc2p in K. lactis oxidative stress response.

A Single Amino Acid Substitution in Structural Protein VP2 Abrogates the Neurotropism of Enterovirus A-71 in Mice.

Enterovirus 71 (EV-A71) causes hand, foot, and mouth disease (HFMD) in children and has been associated with neurological complications. With no specific treatment and a monovalent vaccine limited to the Chinese market, HFMD remains a serious public health concern and an economic burden to affected societies. The molecular mechanisms underpinning EV-A71 neurovirulence have yet to be fully elucidated. In this work, we provide experimental evidence that a single amino acid substitution (I to K) at position 149 in structural protein VP2 of a non-mouse-adapted EV-A71 strain completely and specifically abrogated its infectivity in murine motor neuron-like NSC-34 cells. We showed that VP2 I149K mutant was impaired in murine SCARB2-mediated entry step but retained the ability to attach at the cell surface. In vivo, VP2 I149K mutant was fully attenuated in a symptomatic mouse model of progressive limb paralysis. While viral titers in limb muscles were comparable to mice infected with parental wild-type strain, significantly lower viral titers were measured in the spinal cord and brain, with minimal tissue damage, therefore indicating that VP2 I149K mutant is specifically impaired in its ability to invade the central nervous system (CNS). This study highlights the key role of amino acid at position 149 in VP2 in EV-A71 neurovirulence, and lends further support that the EF loop of VP2 represents a potential therapeutic target.

Genetic Diversity Does Not Contribute to Attenuation of HeLa Passaged Wild-Type Yellow Fever Virus Strain French Viscerotropic Virus.

The disease yellow fever was prevented by two live attenuated vaccines, strains 17D and French neurotropic vaccine (FNV), derived by serial passage of wild-type (WT) strains Asibi and French Viscerotropic virus (FVV), respectively. Both 17D and FNV displayed decreased genetic diversity and resistance to the antiviral Ribavirin compared to their WT parental strains, which are thought to contribute to their attenuated phenotypes. Subsequent studies found that only a few passages of WT strain FVV in HeLa cells resulted in an attenuated virus. In the current study, the genome sequence of FVV following five passages in HeLa cells (FVV HeLa p5) was determined through Next Generation Sequencing (NGS) with the aim to investigate the molecular basis of viral attenuation. It was found that WT FVV and FVV HeLa p5 virus differed by five amino acid substitutions: E-D155A, E-K331R, E-I412V, NS2A-T105A, and NS4B-V98I. Surprisingly, the genetic diversity and Ribavirin resistance of the FVV HeLa p5 virus were not statistically different to WT parent FVV. These findings suggest that while FVV HeLa p5 is attenuated, this is not dependent on a high-fidelity replication complex, characterized by reduced genetic diversity or increased Ribavirin stability, as seen with FNV and 17D vaccines.

The Chlamydia trachomatis inclusion membrane protein CT006 associates with lipid droplets in eukaryotic cells.

Chlamydia trachomatis causes genital and ocular infections in humans. This bacterial pathogen multiplies exclusively within host cells in a characteristic vacuole (inclusion) and delivers proteins such as inclusion membrane proteins (Incs) into the host cell. Here, we identified CT006 as a novel C. trachomatis protein that when expressed ectopically eukaryotic cells can associate with lipid droplets (LDs). A screen using Saccharomyces cerevisiae identified two Incs causing vacuolar protein sorting defects and seven Incs showing tropism for eukaryotic organelles. Ectopic expression in yeast and mammalian cells of genes encoding different fragments of CT006 revealed tropism for the endoplasmic reticulum and LDs. We identified a LD-targeting region within the first 88 amino acid residues of CT006, and positively charged residues important for this targeting. Comparing with the parental wild-type strain, cells infected by a newly generated C. trachomatis strain overproducing CT006 with a double hemagglutinin tag showed a slight increase in the area occupied by LDs within the inclusion region. However, we could not correlate this effect with the LD-targeting regions within CT006. We further showed that both the amino and carboxy-terminal regions of CT006, flanking the Inc-characteristic bilobed hydrophobic domain, are exposed to the host cell cytosol during C. trachomatis infection, supporting their availability to interact with host cell targets. Altogether, our data suggest that CT006 might participate in the interaction of LDs with C. trachomatis inclusions.

Expanding the upper-temperature boundary for the microalga Picochlorum sp. (BPE23) by adaptive laboratory evolution.

Closed photobioreactors reach temperatures that reduce microalgal production or even cause culture collapses. Cooling can maintain the temperature within tolerable boundaries, but cooling is energy-intensive and expensive. Thermotolerant microalgal strains can reduce dependence on such cooling. In this study, adaptive laboratory evolution was performed for 390 days to further increase the maximal tolerable temperature for the already thermotolerant microalgae Picochlorum sp. (BPE23). The parental wild-type strain of Picochlorum sp. (BPE23) exhibited a maximum mid-day growth temperature of 47.5°C, whereas the isolated clones grew up to 49°C. At a lower temperature of 40°C, the growth rate and absorption cross-sectional area were similar for the wild-type strain and the evolved clones. Interestingly, the clones showed a 46% increase in cell volume compared to the wild-type strain. The evolved clones with an expanded upper-temperature boundary can be applied for broader temperature control of 1.5°C, without trade-off effects at lower temperatures.

Biocontrol Activity of Nonpathogenic Strains of Fusarium oxysporum: Colonization on the Root Surface to Overcome Nutritional Competition.

Fusarium oxysporum is a soil-borne fungal pathogen that causes vascular wilts in a wide variety of crops. Certain nonpathogenic strains of F. oxysporum are known to protect crops against F. oxysporum pathogens. We assessed the biocontrol activities of nonpathogenic mutants of F. oxysporum ff. spp. melonis and lycopersici generated by disruption of the FOW2 gene, which encodes a Zn(II)2Cys6-type transcriptional regulator essential for their pathogenicity. Pre-inoculation of melon or tomato roots with strain ΔFOW2 conidia markedly reduced disease incidence caused by the parental wild-type strain in a concentration-dependent manner of conidial suspensions of ΔFOW2 strains. The biocontrol effect caused by the ΔFOW2 pre-inoculation lasted for at least 7 days. Pre-inoculation of melon roots with the wild-type or ΔFOW2 strain of F. oxysporum f. sp. lycopersici and nonpathogenic F. oxysporum strain also led to biocontrol activity against F. oxysporum f. sp. melonis, indicating that the biocontrol activity of ΔFOW2 strains is due to its nonpathogenic nature, not to the FOW2 disfunction. Conidial germination and hyphal elongation of only the wild-type strain were inhibited on melon root surface pre-inoculated with conidia of strains nonpathogenic to melon plants. Expression of defense-related genes was not significantly induced in roots and aboveground parts of melon seedlings preinoculated with ΔFOW2 conidia. Carbon source competition assay showed that nonpathogenic strains competed with the wild-type strain for a carbon source in soil. Strain ΔFOW2 also competed with the oomycete pathogen Pythium aphanidermatum for carbon source and protected melon plants from P. aphanidermatum. Our results suggest that the biocontrol activity of the nonpathogenic F. oxysporum strains used in this study mainly depends on their extensive colonization of the root surface and outcompeting pathogens for nutrients.