The human fungal pathogen Aspergillus fumigatus can produce the highest known number of meiotic crossovers.

Sexual reproduction involving meiosis is essential in most eukaryotes. This produces offspring with novel genotypes, both by segregation of parental chromosomes as well as crossovers between homologous chromosomes. A sexual cycle for the opportunistic human pathogenic fungus Aspergillus fumigatus is known, but the genetic consequences of meiosis have remained unknown. Among other Aspergilli, it is known that A. flavus has a moderately high recombination rate with an average of 4.2 crossovers per chromosome pair, whereas A. nidulans has in contrast a higher rate with 9.3 crossovers per chromosome pair. Here, we show in a cross between A. fumigatus strains that they produce an average of 29.9 crossovers per chromosome pair and large variation in total map length across additional strain crosses. This rate of crossovers per chromosome is more than twice that seen for any known organism, which we discuss in relation to other genetic model systems. We validate this high rate of crossovers through mapping of resistance to the laboratory antifungal acriflavine by using standing variation in an undescribed ABC efflux transporter. We then demonstrate that this rate of crossovers is sufficient to produce one of the common multidrug resistant haplotypes found in the cyp51A gene (TR34/L98H) in crosses among parents harboring either of 2 nearby genetic variants, possibly explaining the early spread of such haplotypes. Our results suggest that genomic studies in this species should reassess common assumptions about linkage between genetic regions. The finding of an unparalleled crossover rate in A. fumigatus provides opportunities to understand why these rates are not generally higher in other eukaryotes.

Parallel Genomic Changes Drive Repeated Evolution of Placentas in Live-Bearing Fish.

The evolutionary origin of complex organs challenges empirical study because most organs evolved hundreds of millions of years ago. The placenta of live-bearing fish in the family Poeciliidae represents a unique opportunity to study the evolutionary origin of complex organs, because in this family a placenta evolved at least nine times independently. It is currently unknown whether this repeated evolution is accompanied by similar, repeated, genomic changes in placental species. Here, we compare whole genomes of 26 poeciliid species representing six out of nine independent origins of placentation. Evolutionary rate analysis revealed that the evolution of the placenta coincides with convergent shifts in the evolutionary rate of 78 protein-coding genes, mainly observed in transporter- and vesicle-located genes. Furthermore, differences in sequence conservation showed that placental evolution coincided with similar changes in 76 noncoding regulatory elements, occurring primarily around genes that regulate development. The unexpected high occurrence of GATA simple repeats in the regulatory elements suggests an important function for GATA repeats in developmental gene regulation. The distinction in molecular evolution observed, with protein-coding parallel changes more often found in metabolic and structural pathways, compared with regulatory change more frequently found in developmental pathways, offers a compelling model for complex trait evolution in general: changing the regulation of otherwise highly conserved developmental genes may allow for the evolution of complex traits.

Dietary Inulin Increases Lactiplantibacillus plantarum Strain Lp900 Persistence in Rats Depending on the Dietary-Calcium Level.

Synbiotics are food supplements that combine probiotics and prebiotics to synergistically elicit health benefits in the consumer. Lactiplantibacillus plantarum strains display high survival during transit through the mammalian gastrointestinal tract and were shown to have health-promoting properties. Growth on the fructose polysaccharide inulin is relatively uncommon in L. plantarum, and in this study we describe FosE, a plasmid-encoded ß-fructosidase of L. plantarum strain Lp900 which has inulin-hydrolyzing properties. FosE contains an LPxTG-like motif involved in sortase-dependent cell wall anchoring but is also (partially) released in the culture supernatant. In addition, we examined the effect of diet supplementation with inulin on the intestinal persistence of Lp900 in adult male Wistar rats in diets with distinct calcium levels. Inulin supplementation in high-dietary-calcium diets significantly increased the intestinal persistence of L. plantarum Lp900, whereas this effect was not observed upon inulin supplementation of the low-calcium diet. Moreover, intestinal persistence of L. plantarum Lp900 was determined when provided as a probiotic (by itself) or as a synbiotic (i.e., in an inulin suspension) in rats that were fed unsupplemented diets containing the different calcium levels, revealing that the synbiotic administration increased bacterial survival and led to higher abundance of L. plantarum Lp900 in rats, particularly in a low-calcium-diet context. Our findings demonstrate that inulin supplementation can significantly enhance the intestinal delivery of L. plantarum Lp900 but that this effect strongly depends on calcium levels in the diet.IMPORTANCE Synbiotics combine probiotics with prebiotics to synergistically elicit a health benefit in the consumer. Previous studies have shown that prebiotics can selectively stimulate the growth in the intestine of specific bacterial strains. In synbiotic supplementations the prebiotics constituent could increase the intestinal persistence and survival of accompanying probiotic strain(s) and/or modulate the endogenous host microbiota to contribute to the synergistic enhancement of the health-promoting effects of the synbiotic constituents. Our study establishes a profound effect of dietary-calcium-dependent inulin supplementation on the intestinal persistence of inulin-utilizing L. plantarum Lp900 in rats. We also show that in rats on a low-dietary-calcium regime, the survival and intestinal abundance of L. plantarum Lp900 are significantly increased by administering it as an inulin-containing synbiotic. This study demonstrates that prebiotics can enhance the intestinal delivery of specific probiotics and that the prebiotic effect is profoundly influenced by the calcium content of the diet.

Biases in bulk: DNA metabarcoding of marine communities and the methodology involved.

With the growing anthropogenic pressure on marine ecosystems, the need for efficient monitoring of biodiversity grows stronger. DNA metabarcoding of bulk samples is increasingly being implemented in ecosystem assessments and is more cost-efficient and less time-consuming than monitoring based on morphology. However, before raw sequences are obtained from bulk samples, a profound number of methodological choices must be made. Here, we critically review the recent methods used for metabarcoding of marine bulk samples (including benthic, plankton and diet samples) and indicate how potential biases can be introduced throughout sampling, preprocessing, DNA extraction, marker and primer selection, PCR amplification and sequencing. From a total of 64 studies evaluated, our recommendations for best practices include to (a) consider DESS as a fixative instead of ethanol, (b) use the DNeasy PowerSoil kit for any samples containing traces of sediment, (c) not limit the marker selection to COI only, but preferably include multiple markers for higher taxonomic resolution, (d) avoid touchdown PCR profiles, (e) use a fixed annealing temperature for each primer pair when comparing across studies or institutes, (f) use a minimum of three PCR replicates, and (g) include both negative and positive controls. Although the implementation of DNA metabarcoding still faces several technical complexities, we foresee wide-ranging advances in the near future, including improved bioinformatics for taxonomic assignment, sequencing of longer fragments and the use of whole-genome information. Despite the bulk of biases involved in metabarcoding of bulk samples, if appropriate controls are included along the data generation process, it is clear that DNA metabarcoding provides a valuable tool in ecosystem assessments.

Trade-offs between reducing complex terminology and producing accurate interpretations from environmental DNA: Comment on "Environmental DNA: What's behind the term?" by Pawlowski et al., (2020).

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Immune evasion by a staphylococcal inhibitor of myeloperoxidase.

Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.

The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility.

Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors are essential for initiating and propagating the eukaryotic innate immune signaling cascade. Here, we investigate TirS, a Staphylococcus aureus TIR mimic that is part of a novel bacterial invasion mechanism. Its ectopic expression in eukaryotic cells inhibited TLR signaling, downregulating the NF-kB pathway through inhibition of TLR2, TLR4, TLR5, and TLR9. Skin lesions induced by the S. aureus knockout tirS mutant increased in a mouse model compared with wild-type and restored strains even though the tirS-mutant and wild-type strains did not differ in bacterial load. TirS also was associated with lower neutrophil and macrophage activity, confirming a central role in virulence attenuation through local inflammatory responses. TirS invariably localizes within the staphylococcal chromosomal cassettes (SCC) containing the fusC gene for fusidic acid resistance but not always carrying the mecA gene. Of note, sub-inhibitory concentration of fusidic acid increased tirS expression. Epidemiological studies identified no link between this effector and clinical presentation but showed a selective advantage with a SCCmec element with SCC fusC/tirS. Thus, two key traits determining the success and spread of bacterial infections are linked.

Correction: The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility.

[This corrects the article DOI: 10.1371/journal.ppat.1006092.].

Neutrophils versus Staphylococcus aureus: a biological tug of war.

The pathogen Staphylococcus aureus is well adapted to its human host. Neutrophil-mediated killing is a crucial defense system against S. aureus; however, the pathogen has evolved many strategies to resist killing. We first describe the discrete steps of neutrophil activation and migration to the site of infection and the killing of microbes by neutrophils in general. We then highlight the different approaches utilized by S. aureus to resist the different steps of neutrophil attack. Various molecules are discussed in their evolutionary context. Most of the molecules secreted by S. aureus to combat neutrophil attacks at the site of infection show clear human specificity. Many elements of human neutrophil defenses appear redundant, and so the evasion strategies of staphylococci display redundant functions as well. All efforts by S. aureus to resist neutrophil-mediated killing stress the importance of these mechanisms in the pathophysiology of staphylococcal diseases. However, the highly human-specific nature of most host-pathogen interactions hinders the in vivo establishment of their contribution to staphylococcal pathophysiology.

Bright fluorescent Streptococcus pneumoniae for live-cell imaging of host-pathogen interactions.

Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis.

The meningococcal autotransporter AutA is implicated in autoaggregation and biofilm formation.

Autotransporters (ATs) are proteins secreted by Gram-negative bacteria that often play a role in virulence. Eight different ATs have been identified in Neisseria meningitidis, but only six of them have been characterized. AutA is one of the remaining ATs. Its expression remains controversial. Here, we show that the autA gene is present in many neisserial species, but its expression is often disrupted by various genetic features; however, it is expressed in certain strains of N. meningitidis. By sequencing the autA gene in large panels of disease isolates and Western blot analysis, we demonstrated that AutA expression is prone to phase variation at AAGC nucleotide repeats located within the DNA encoding the signal sequence. AutA is not secreted into the extracellular medium, but remains associated with the bacterial cell surface. We further demonstrate that AutA expression induces autoaggregation in a process that, dependent on the particular strain, may require extracellular DNA (eDNA). This property influences the organization of bacterial communities like lattices and biofilms. In vitro assays evidenced that AutA is a self-associating AT that binds DNA. We suggest that AutA-mediated autoaggregation might be particularly important for colonization and persistence of the pathogen in the nasopharynx of the host.

Involvement of three meningococcal surface-exposed proteins, the heparin-binding protein NhbA, the α-peptide of IgA protease and the autotransporter protease NalP, in initiation of biofilm formation.

Neisseria meningitidis is a common and usually harmless inhabitant of the mucosa of the human nasopharynx, which, in rare cases, can cross the epithelial barrier and cause meningitis and sepsis. Biofilm formation favours the colonization of the host and the subsequent carrier state. Two different strategies of biofilm formation, either dependent or independent on extracellular DNA (eDNA), have been described for meningococcal strains. Here, we demonstrate that the autotransporter protease NalP, the expression of which is phase variable, affects eDNA-dependent biofilm formation in N. meningitidis. The effect of NalP was found in biofilm formation under static and flow conditions and was dependent on its protease activity. Cleavage of the heparin-binding antigen NhbA and the α-peptide of IgA protease, resulting in the release of positively charged polypeptides from the cell surface, was responsible for the reduction in biofilm formation when NalP is expressed. Both NhbA and the α-peptide of IgA protease were shown to bind DNA. We conclude that NhbA and the α-peptide of IgA protease are implicated in biofilm formation by binding eDNA and that NalP is an important regulator of this process through the proteolysis of these surface-exposed proteins.

Inactivation of staphylococcal phenol soluble modulins by serum lipoprotein particles.

Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered.

Distinct localization of the complement C5b-9 complex on Gram-positive bacteria.

The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo-attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b-9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram-positive bacteria are protected from MAC-dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram-positive pathogens secrete small proteins that inhibit C5b-9 formation. In this study, we found that complement activation on Gram-positive bacteria in serum results in specific surface deposition of C5b-9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS-stable) forms, indicating the presence of ring-structured C5b-9. Surprisingly, confocal microscopy demonstrated that C5b-9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b-9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b-9 complex and Gram-positive bacteria, which might ultimately lead to a new model of MAC assembly and functioning.

Recognition of LPS by TLR4: potential for anti-inflammatory therapies.

LPS molecules of marine bacteria show structures distinct from terrestrial bacteria, due to the different environment that marine bacteria live in. Because of these different structures, lipid A molecules from marine bacteria are most often poor stimulators of the Toll-like receptor 4 (TLR4) pathway. Due to their low stimulatory potential, these lipid A molecules are suggested to be applicable as antagonists of TLR4 signaling in sepsis patients, where this immune response is amplified and unregulated. Antagonizing lipid A molecules might be used for future therapies against sepsis, therapies that currently do not exist. In this review, we will discuss these differences in lipid A structures and their recognition by the immune system. The modifications present in marine lipid A structures are described, and their potential as LPS antagonists will be discussed. Finally, since clinical trials built on antagonizing lipid A molecules have proven unsuccessful, we propose to also focus on different aspects of the TLR4 signaling pathway when searching for new potential drugs. Furthermore, we put forward the notion that bacteria probably already produce inhibitors of TLR4 signaling, making these bacterial products interesting molecules to investigate for future sepsis therapies.

eDNA based bycatch assessment in pelagic fish catches.

Pelagic fish like herring, sardines, and mackerel constitute an essential and nutritious human food source globally. Their sustainable harvest is promoted by the application of precise, accurate, and cost-effective methods for estimating bycatch. Here, we experimentally test the new concept of using eDNA for quantitative bycatch assessment on the illustrative example of the Baltic Sea sprat fisheries with herring bycatch. We investigate the full pipeline from sampling of production water on vessels and in processing factories to the estimation of species weight fractions. Using a series of controlled mixture experiments, we demonstrate that the eDNA signal from production water shows a strong, seasonally consistent linear relationship with herring weight fractions, however, the relationship is influenced by the molecular method used (qPCR or metabarcoding). In four large sprat landings analyzed, despite examples of remarkable consistency between eDNA and visual reporting, estimates of herring bycatch biomass varied between the methods applied, with the eDNA-based estimates having the highest precision for all landings analyzed. The eDNA-based bycatch assessment method has the potential to improve the quality and cost effectiveness of bycatch assessment in large pelagic fisheries catches and in the long run lead to more sustainable management of pelagic fish as a precious marine resource.

Edge computing in wildlife behavior and ecology.

Modern sensor technologies increasingly enrich studies in wildlife behavior and ecology. However, constraints on weight, connectivity, energy and memory availability limit their implementation. With the advent of edge computing, there is increasing potential to mitigate these constraints, and drive major advancements in wildlife studies.

Staphylococcus aureus elaborates leukocidin AB to mediate escape from within human neutrophils.

Methicillin-resistant Staphylococcus aureus (MRSA) strains of the pulsed-field type USA300 are primarily responsible for the current community-associated epidemic of MRSA infections in the United States. The success of USA300 is partly attributed to the ability of the pathogen to avoid destruction by human neutrophils (polymorphonuclear leukocytes [PMNs]), which are crucial to the host immune response to S. aureus infection. In this work, we investigated the contribution of bicomponent pore-forming toxins to the ability of USA300 to withstand attack from primary human PMNs. We demonstrate that in vitro growth conditions influence the expression, production, and availability of leukotoxins by USA300, which in turn impact the cytotoxic potential of this clone toward PMNs. Interestingly, we also found that upon exposure to PMNs, USA300 preferentially activates the promoter of the lukAB operon, which encodes the recently identified leukocidin AB (LukAB). LukAB elaborated by extracellular S. aureus forms pores in the plasma membrane of PMNs, leading to PMN lysis, highlighting a contribution of LukAB to USA300 virulence. We now show that LukAB also facilitates the escape of bacteria engulfed within PMNs, in turn enabling the replication and outgrowth of S. aureus. Together, these results suggest that upon encountering PMNs S. aureus induces the production of LukAB, which serves as an extra- and intracellular weapon to protect the bacterium from destruction by human PMNs.

baseLess: lightweight detection of sequences in raw MinION data.

Summary: With its candybar form factor and low initial investment cost, the MinION brought affordable portable nucleic acid analysis within reach. However, translating the electrical signal it outputs into a sequence of bases still requires mid-tier computer hardware, which remains a caveat when aiming for deployment of many devices at once or usage in remote areas. For applications focusing on detection of a target sequence, such as infectious disease monitoring or species identification, the computational cost of analysis may be reduced by directly detecting the target sequence in the electrical signal instead. Here, we present baseLess, a computational tool that enables such target-detection-only analysis. BaseLess makes use of an array of small neural networks, each of which efficiently detects a fixed-size subsequence of the target sequence directly from the electrical signal. We show that baseLess can accurately determine the identity of reads between three closely related fish species and can classify sequences in mixtures of 20 bacterial species, on an inexpensive single-board computer. Availability and implementation: baseLess and all code used in data preparation and validation are available on Github at https://github.com/cvdelannoy/baseLess, under an MIT license. Used validation data and scripts can be found at https://doi.org/10.4121/20261392, under an MIT license. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

A closed Candidatus Odinarchaeum chromosome exposes Asgard archaeal viruses.

Asgard archaea have recently been identified as the closest archaeal relatives of eukaryotes. Their ecology, and particularly their virome, remain enigmatic. We reassembled and closed the chromosome of Candidatus Odinarchaeum yellowstonii LCB_4, through long-range PCR, revealing CRISPR spacers targeting viral contigs. We found related viruses in the genomes of diverse prokaryotes from geothermal environments, including other Asgard archaea. These viruses open research avenues into the ecology and evolution of Asgard archaea.