Host-specific Adaptation Research Articles

Conservation and Expansion of Transcriptional Factor Repertoire in the Fusarium oxysporum Species Complex.

The Fusarium oxysporum species complex (FOSC) includes both plant and human pathogens that cause devastating plant vascular wilt diseases and threaten public health. Each F. oxysporum genome comprises core chromosomes (CCs) for housekeeping functions and accessory chromosomes (ACs) that contribute to host-specific adaptation. This study inspects global transcription factor profiles (TFomes) and their potential roles in coordinating CC and AC functions to accomplish host-specific interactions. Remarkably, we found a clear positive correlation between the sizes of TFomes and the proteomes of an organism. With the acquisition of ACs, the FOSC TFomes were larger than the other fungal genomes included in this study. Among a total of 48 classified TF families, 14 families involved in transcription/translation regulations and cell cycle controls were highly conserved. Among the 30 FOSC expanded families, Zn2-C6 and Znf_C2H2 were most significantly expanded to 671 and 167 genes per family including well-characterized homologs of Ftf1 (Zn2-C6) and PacC (Znf_C2H2) that are involved in host-specific interactions. Manual curation of characterized TFs increased the TFome repertoires by 3% including a disordered protein Ren1. RNA-Seq revealed a steady pattern of expression for conserved TF families and specific activation for AC TFs. Functional characterization of these TFs could enhance our understanding of transcriptional regulation involved in FOSC cross-kingdom interactions, disentangle species-specific adaptation, and identify targets to combat diverse diseases caused by this group of fungal pathogens.

Host specific adaptations of Ligilactobacillus aviarius to poultry

The genus Ligilactobacillus encompasses species adapted to vertebrate hosts and fermented food. Their genomes encode adaptations to the host lifestyle. Reports of gut microbiota from chicken and turkey gastrointestinal tract have shown a high persistence of Ligilactobacillus aviarius along the digestive system compared to other species found in the same host. However, its adaptations to poultry as a host has not yet been described. In this work, the pan-genome of Ligilactobacillus aviarius was explored to describe the functional adaptability to the gastrointestinal environment. The core genome is composed of 1179 gene clusters that are present at least in one copy that codifies to structural, ribosomal and biogenesis proteins. The rest of the identified regions were classified into three different functional clusters of orthologous groups (clusters) that codify carbohydrate metabolism, envelope biogenesis, viral defence mechanisms, and mobilome inclusions. The pan-genome of Ligilactobacillus aviarius is a closed pan-genome, frequently found in poultry and highly prevalent across chicken faecal samples. The genome of L. aviarius codifies different clusters of glycoside hydrolases and glycosyltransferases that mediate interactions with the host cells. Accessory features, such as antiviral mechanisms and prophage inclusions, variate amongst strains from different GIT sections. This information provides hints about the interaction of this species with viral particles and other bacterial species. This work highlights functional adaptability traits present in L. aviarius that make it a dominant key member of the poultry gut microbiota and enlightens the convergent ecological relation of this species to the poultry gut environment.

Investigation and sequence analysis of psittacine beak and feather disease virus and avian polyomavirus from companion birds in Windhoek, Namibia

The commercial farming and trading of parrots and ornamental birds as companion animals are important economic activities in many countries. Some of the bird species farmed/traded are captured from the wild or are closely related to wild birds and therefore represent a risk of pathogen exchange/introduction. Beak and feather disease virus (BFDV) and avian poliomavirus (APV) are among the viruses with the biggest impact on companion bird populations and have been detected in different hosts worldwide. Despite their relevance for both domesticated and wild birds, our knowledge of BFDV and APV epidemiology remains limited in several African countries. In the present study, 143 cloacal swabs were collected from companion birds in Windhoek, Namibia, and tested by polymerase chain reaction for BFDV and APV. Of the samples tested, 35/143 (24.48%) tested positive for BFDV; 11/143 (7.69%) were positive for APV; and 6/143 (4.2%) tested positive for both pathogens. Positive amplicons, consisting of segments of the ORF1 and VP1 genes, were sequenced and compared with sequences from viruses identified in other countries. Four Namibian-only clades of BFDV were identified, loosely related to foreign strains, which suggest the occurrence of multiple introduction events in the past, potentially from South Africa, followed by local, independent evolution. In contrast, the Namibian APV sequences were identical to each other and form a single clade. In both instances, no correlation was observed between the sampling host and the viral phylogeny, suggesting the absence of host-specific adaptation and a remarkable, unconstrained viral circulation within Namibian borders. Therefore, while regulations and control measures developed against foreign strain introduction have proven to be effective over time, the spread of BFDV and APV within Namibia's borders appears undeterred. Additional resources should be dedicated to limit strain circulation in commercial farming facilities, markets and small-scale traders.

Stem-Loop I of the Tembusu Virus 3'-Untranslated Region Is Responsible for Viral Host-Specific Adaptation and the Pathogenicity of the Virus in Mice.

ABSTRACTTembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3′-untranslated region (3′UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3′UTRs. Compared with rMM 1775, rMM-CQ3′UTR (recombinant MM 1775 virus carrying the 3′UTR of CQW1) exhibited enhanced proliferation in vitro, with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3′UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads. In contrast, rCQ-MM3′UTR showed similar growth kinetics in vitro and neurovirulence in mice compared with those of rCQW1. Then, the Stem-loop I (SLI) structure, which showed the highest variation within the 3′UTR between CQW1 and MM 1775, was further chosen for making chimeric viruses. The peak titers of rMM-CQ3′UTRSLI displayed a 15- or 4-fold increase in vitro, and the neurovirulence in mice was attenuated, compared with that of rMM 1775; rCQ-MM3′UTRSLI displayed comparable multiplication ability in vitro but was significantly attenuated in mice, in contrast with rCQW1. In conclusion, we demonstrated that the TMUV SLI structure of the 3′UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old mice, providing a new understanding of the functions of TMUV 3′UTR in viral host switching and the pathogenicity changes in mice.IMPORTANCE Mosquito-borne flaviviruses (MBFVs) constitute a large number of mosquito-transmitted viruses. The 3′-untranslated region (3′UTR) of MBFV has been suggested to be relevant to viral host-specific adaptation. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3′UTR are largely unknown. Here, using Tembusu virus (TMUV), an avian MBFV as models, we observed that the duck-derived SLI of the 3′UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3′UTR in viral host switching and pathogenicity changes in mice.

Strain-level profiling with picodroplet microfluidic cultivation reveals host-specific adaption of honeybee gut symbionts

BackgroundSymbiotic gut microbes have a rich genomic and metabolic pool and are closely related to hosts’ health. Traditional sequencing profiling masks the genomic and phenotypic diversity among strains from the same species. Innovative droplet-based microfluidic cultivation may help to elucidate the inter-strain interactions. A limited number of bacterial phylotypes colonize the honeybee gut, while individual strains possess unique genomic potential and critical capabilities, which provides a particularly good model for strain-level analyses.ResultsHere, we construct a droplet-based microfluidic platform and generated ~ 6 × 108 droplets encapsulated with individual bacterial cells from the honeybee gut and cultivate in different media. Shotgun metagenomic analysis reveals significant changes in community structure after droplet-based cultivation, with certain species showing higher strain-level diversity than in gut samples. We obtain metagenome-assembled genomes, and comparative analysis reveal a potential novel cluster from Bifidobacterium in the honeybee. Interestingly, Lactobacillus panisapium strains obtained via droplet cultivation from Apis mellifera contain a unique set of genes encoding l-arabinofuranosidase, which is likely important for the survival of bacteria in competitive environments.ConclusionsBy encapsulating single bacteria cells inside microfluidic droplets, we exclude potential interspecific competition for the enrichment of rare strains by shotgun sequencing at high resolution. The comparative genomic analysis reveals underlying mechanisms for host-specific adaptations, providing intriguing insights into microbe-microbe interactions. The current approach may facilitate the hunting for elusive bacteria and paves the way for large-scale studies of more complex animal microbial communities.6MWJv9N2gCCgq2b2j_Wic-Video

Predictive modeling of Pseudomonas syringae virulence on bean using gradient boosted decision trees.

Pseudomonas syringae is a genetically diverse bacterial species complex responsible for numerous agronomically important crop diseases. Individual P. syringae isolates are assigned pathovar designations based on their host of isolation and the associated disease symptoms, and these pathovar designations are often assumed to reflect host specificity although this assumption has rarely been rigorously tested. Here we developed a rapid seed infection assay to measure the virulence of 121 diverse P. syringae isolates on common bean (Phaseolus vulgaris). This collection includes P. syringae phylogroup 2 (PG2) bean isolates (pathovar syringae) that cause bacterial spot disease and P. syringae phylogroup 3 (PG3) bean isolates (pathovar phaseolicola) that cause the more serious halo blight disease. We found that bean isolates in general were significantly more virulent on bean than non-bean isolates and observed no significant virulence difference between the PG2 and PG3 bean isolates. However, when we compared virulence within PGs we found that PG3 bean isolates were significantly more virulent than PG3 non-bean isolates, while there was no significant difference in virulence between PG2 bean and non-bean isolates. These results indicate that PG3 strains have a higher level of host specificity than PG2 strains. We then used gradient boosting machine learning to predict each strain’s virulence on bean based on whole genome k-mers, type III secreted effector k-mers, and the presence/absence of type III effectors and phytotoxins. Our model performed best using whole genome data and was able to predict virulence with high accuracy (mean absolute error = 0.05). Finally, we functionally validated the model by predicting virulence for 16 strains and found that 15 (94%) had virulence levels within the bounds of estimated predictions. This study strengthens the hypothesis that P. syringae PG2 strains have evolved a different lifestyle than other P. syringae strains as reflected in their lower level of host specificity. It also acts as a proof-of-principle to demonstrate the power of machine learning for predicting host specific adaptation.

Enterovirus A Shows Unique Patterns of Codon Usage Bias in Conventional Versus Unconventional Clade.

Enterovirus A (EV-A) species cause hand, foot and mouth disease (HFMD), threatening the health of young children. Understanding the mutual codon usage pattern of the virus and its host(s) has fundamental and applied values. Here, through examining multiple codon usage parameters, we found that the codon usage bias among EV-A strains varies and is clade-specific. EVA76, EVA89, EVA90, EVA91 and EVA92, the unconventional clade of EV-A strains, show unique codon usage pattern relative to the two conventional clades, including EVA71, CVA16, CVA6 and CVA10, etc. Analyses of Effective Number of Codon (ENC), Correspondence Analysis (COA) and Parity Rule 2 (PR2), etc., revealed that the codon usage patterns of EV-A strains are shaped by mutation pressure and natural selection. Based on the neutrality analysis, we determined the dominant role of natural selection in the formation of the codon usage bias of EV-A. In addition, we have determined the codon usage compatibility of potential hosts for EV-A strains using codon adaptation index (CAI), relative codon deoptimization index (RCDI) and similarity index (SiD) analyses, and found that EV-A showed host-specific codon adaptation patterns in different clades. Finally, we confirmed that the unique codon usage pattern of the unconventional clade affected protein expression level in human cell lines. In conclusion, we identified novel characteristics of codon usage bias in distinct EV-A clades associated with their host range, transmission and pathogenicity.

Design of typical genes for heterologous gene expression

Heterologous protein expression is an important method for analysing cellular functions of proteins, in genetic circuit engineering and in overexpressing proteins for biopharmaceutical applications and structural biology research. The degeneracy of the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, plays an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the influence of a profiled codon usage adaptation approach on protein expression levels in the eukaryotic model organism Saccharomyces cerevisiae. We selected green fluorescent protein (GFP) and human α-synuclein (αSyn) as representatives for stable and intrinsically disordered proteins and representing a benchmark and a challenging test case. A new approach was implemented to design typical genes resembling the codon usage of any subset of endogenous genes. Using this approach, synthetic genes for GFP and αSyn were generated, heterologously expressed and evaluated in yeast. We demonstrate that GFP is expressed at high levels, and that the toxic αSyn can be adapted to endogenous, low-level expression. The new software is publicly available as a web-application for performing host-specific protein adaptations to a set of the most commonly used model organisms (https://odysseus.motorprotein.de).

Reduced IFN-ß inhibitory activity of Lagos bat virus phosphoproteins in human compared to Eidolon helvum bat cells.

Eidolon helvum bats are reservoir hosts for highly pathogenic lyssaviruses often showing limited disease upon natural infection. An enhanced antiviral interferon (IFN) response combined with reduced inflammation might be linked to the apparent virus tolerance in bats. Lyssavirus phosphoproteins inhibit the IFN response with virus strain-specific efficiency. To date, little is known regarding the lyssavirus P-dependent anti-IFN countermeasures in bats, mainly due to a lack of in vitro tools. By using E. helvum bat cell cultures in a newly established bat-specific IFN-promoter activation assay, we analyzed the IFN-ß inhibitory activity of multiple lyssavirus P in E. helvum compared to human cells. Initial virus infection studies with a recently isolated E. helvum-borne Lagos bat virus street strain from Ghana showed enhanced LBV propagation in an E. helvum lung cell line compared to human A549 lung cells at later time points suggesting effective viral countermeasures against cellular defense mechanisms. A direct comparison of the IFN-ß inhibitory activity of the LBV-GH P protein with other lyssavirus P proteins showed that LBV-GH P and RVP both strongly inhibited the bat IFN-β promotor activation (range 75-90%) in EidLu/20.2 and an E. helvum kidney cell line. Conversely, LBV-GH P blocked the activation of the human IFN-β promoter less efficiently compared to a prototypic Rabies virus P protein (range LBV P 52-68% vs RVP 71-95%) in two different human cell lines (HEK-293T, A549). The same pattern was seen for two prototypic LBV P variants suggesting an overall reduced LBV P IFN-ß inhibitory activity in human cells as compared to E. helvum bat cells. Increased IFN-ß inhibition by lyssavirus P in reservoir host cells might be a result of host-specific adaptation processes towards an enhanced IFN response in bat cells.

Long-Read Sequencing Reveals Genetic Adaptation of Bartonella Adhesin A Among Different Bartonella henselae Isolates.

Bartonella henselae is the causative agent of cat scratch disease and other clinical entities such as endocarditis and bacillary angiomatosis. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations. Human, feline, and laboratory adapted B. henselae isolates often display genomic and phenotypic differences that are related to the expression of outer membrane proteins, for example the Bartonella adhesin A (BadA). This modularly-structured trimeric autotransporter adhesin is a major virulence factor of B. henselae and is crucial for the initial binding to the host via the extracellular matrix proteins fibronectin and collagen. By using next-generation long-read sequencing we demonstrate a conserved genome among eight B. henselae isolates and identify a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes. Two of the eight tested B. henselae strains lack BadA expression because of frameshift mutations. We suggest that active recombination mechanisms, possibly via phase variation (i.e., slipped-strand mispairing and site-specific recombination) within the repetitive badA island facilitate reshuffling of homologous domain arrays. The resulting variations among the different BadA proteins might contribute to host immune evasion and enhance long-term and efficient colonisation in the differing host environments. Considering the role of BadA as a key virulence factor, it remains important to check consistently and regularly for BadA surface expression during experimental infection procedures.

Deep sequencing across multiple host species tests pine-endophyte specificity.

Foliar fungal endophytes vary in their distributions across landscapes or plant host taxa, indicative of specialized ecologies and host specific adaptations. Accounts of specialization, however, depend on the taxonomic breadth and geographic range of the host plants included in each study. A broad region-scale study or deep sampling of diverse potential host species still remains relatively rare but is becoming increasingly possible with high-throughput sequencing. Amplicon sequencing was used to rapidly identify the fungal endophytic community among six pine (Pinus, Pinaceae) species co-occurring across northeastern United States and to test for site and host specialization. We focused on the endophytic genus Lophodermium (Rhytismataceae), whose species members are thought to specialize on different pine species, to test if amplicon sequencing could rapidly verify previously implied or discover new patterns of host specificity. While amplicon sequencing could analyze more samples at greater depths and recover greater numbers of unique Lophodermium taxa than when endophyte communities were surveyed with traditional culturing methods, patterns of specialization were not better supported. This may be because amplicon sequencing can indiscriminately capture non-host specific organisms found incidentally from plant tissues or because we have overestimated host-specificity in the past with biased culturing techniques. Amplicon sequencing can quickly identify patterns of host specificity by allowing large-scale surveys but has limitations in quantifying the level of intimacy of these relationships.

Number of Candidate Effector Genes in Accessory Genomes Differentiates Pathogenic From Endophytic Fusarium oxysporum Strains.

The fungus Fusarium oxysporum (Fo) is widely known for causing wilt disease in over 100 different plant species. Endophytic interactions of Fo with plants are much more common, and strains pathogenic on one plant species can even be beneficial endophytes on another species. However, endophytic and beneficial interactions have been much less investigated at the molecular level, and the genetic basis that underlies endophytic versus pathogenic behavior is unknown. To investigate this, 44 Fo strains from non-cultivated Australian soils, grass roots from Spain, and tomato stems from United States were characterized genotypically by whole genome sequencing, and phenotypically by examining their ability to symptomlessly colonize tomato plants and to confer resistance against Fusarium Wilt. Comparison of the genomes of the validated endophytic Fo strains with those of 102 pathogenic strains revealed that both groups have similar genomes sizes, with similar amount of accessory DNA. However, although endophytic strains can harbor homologs of known effector genes, they have typically fewer effector gene candidates and associated non-autonomous transposons (mimps) than pathogenic strains. A pathogenic ‘lifestyle’ is associated with extended effector gene catalogs and a set of “host specific” effectors. No candidate effector genes unique to endophytic strains isolated from the same plant species were found, implying little or no host-specific adaptation. As plant-beneficial interactions were observed to be common for the tested Fo isolates, the propensity for endophytism and the ability to confer biocontrol appears to be a predominant feature of this organism. These findings allow prediction of the lifestyle of a Fo strain based on its genome sequence as a potential pathogen or as a harmless or even beneficial endophyte by determining its effectorome and mimp number.

Host Colonization as a Major Evolutionary Force Favoring the Diversity and the Emergence of the Worldwide Multidrug-Resistant Escherichia coli ST131.

ABSTRACTThe emergence of multidrug-resistant Escherichia coli ST131 is a major worldwide public health problem in humans. According to the “one health” approach, this study investigated animal reservoirs of ST131, their relationships with human strains, and the genetic features associated with host colonization. High-quality genomes originating from human, avian, and canine hosts were classified on the basis of their accessory gene content using pangenomic. Pangenomic clusters and subclusters were specifically and significantly associated with hosts. The functions of clustering accessory genes were mainly enriched in functions involved in DNA acquisition, interactions, and virulence (e.g., pathogenesis, response to biotic stimulus and interaction between organisms). Accordingly, networks of cooccurrent host interaction factors were significantly associated with the pangenomic clusters and the originating hosts. The avian strains exhibited a specific content in virulence factors. Rarely found in humans, they corresponded to pathovars responsible for severe human infections. An emerging subcluster significantly associated with both human and canine hosts was evidenced. This ability to significantly colonize canine hosts in addition to humans was associated with a specific content in virulence factors (VFs) and metabolic functions encoded by a new pathogenicity island in ST131 and an improved fitness that is probably involved in its emergence. Overall, VF content, unlike the determinants of antimicrobial resistance, appeared as a key actor of bacterial host adaptation. The host dimension emerges as a major driver of genetic evolution that shapes ST131 genome, enhances its diversity, and favors its dissemination.

Analysis of Single Nucleotide Variants (SNVs) Induced by Passages of Equine Influenza Virus H3N8 in Embryonated Chicken Eggs.

Vaccination is an effective method for the prevention of influenza virus infection. Many manufacturers use embryonated chicken eggs (ECE) for the propagation of vaccine strains. However, the adaptation of viral strains during subsequent passages can lead to additional virus evolution and lower effectiveness of the resulting vaccines. In our study, we analyzed the distribution of single nucleotide variants (SNVs) of equine influenza virus (EIV) during passaging in ECE. Viral RNA from passage 0 (nasal swabs), passage 2 and 5 was sequenced using next generation technology. In total, 50 SNVs with an occurrence frequency above 2% were observed, 29 of which resulted in amino acid changes. The highest variability was found in passage 2, with the most variable segment being IV encoding hemagglutinin (HA). Three variants, HA (W222G), PB2 (A377E) and PA (R531K), had clearly increased frequency with the subsequent passages, becoming dominant. None of the five nonsynonymous HA variants directly affected the major antigenic sites; however, S227P was previously reported to influence the antigenicity of EIV. Our results suggest that although host-specific adaptation was observed in low passages of EIV in ECE, it should not pose a significant risk to influenza vaccine efficacy.

Closely related Lak megaphages replicate in the microbiomes of diverse animals

SummaryLak phages with alternatively coded ∼540 kbp genomes were recently reported to replicate in Prevotella in microbiomes of humans that consume a non-Western diet, baboons, and pigs. Here, we explore Lak phage diversity and broader distribution using diagnostic polymerase chain reaction and genome-resolved metagenomics. Lak phages were detected in 13 animal types, including reptiles, and are particularly prevalent in pigs. Tracking Lak through the pig gastrointestinal tract revealed significant enrichment in the hindgut compared to the foregut. We reconstructed 34 new Lak genomes, including six curated complete genomes, all of which are alternatively coded. An anomalously large (∼660 kbp) complete genome reconstructed for the most deeply branched Lak from a horse microbiome is also alternatively coded. From the Lak genomes, we identified proteins associated with specific animal species; notably, most have no functional predictions. The presence of closely related Lak phages in diverse animals indicates facile distribution coupled to host-specific adaptation.

Proteomic analysis of serial isolates of Trichosporon asahii identifies host-specific adaptations using the TMT/MRM approach

The opportunistic fungal pathogen Trichosporon asahii (T. asahii) is an important causal agent of mortality in immunocompromised patients and associated with frequent relapses, even with sufficient antifungal treatment. Investigating the proteomes of initial and recurrent isolates may help to identify within-host adaptive changes. In this study, using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (LC–MS/MS) technology, we analyzed the proteomes of two T. asahii strains that were isolated 15 years apart from the same patient who suffered initial and recurrent episodes of systemic disseminated trichosporonosis. A total of 597 differentially expressed proteins were identified. Functional analysis showed that the increased proteins were primarily concentrated on peptide/protein/energy/drug metabolism and translation. Most of the results were determined to be consistent with the findings of phenotypic assays, such as tests for drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays. Moreover, we performed multiple reaction monitoring (MRM) mass spectrometry to verify 27 candidate proteins, and the results of this experiment were also highly consistent with the results of the TMT analysis. Therefore, to the best of our knowledge, these data provide the first molecular evidence of how the T. asahii proteome changes related to host-specific adaptation during human infection. SignificanceSystemic infection with Trichosporon asahii (T. asahii) has recently been recognized as an important causal agent of mortality in immunocompromised patients. Although triazole treatment usually works efficiently in the early phase of infection, many patients relapse. Hence, comparative analyses of the proteomics of initial and recurrent isolates may reveal evidence of adaptive changes within the host. Our study demonstrates that the recurrent strain has undergone proteomic changes using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (LC–MS/MS). Moreover, the results of phenotypic assays, including drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays, were highly consistent with the proteomic changes, and multiple reaction monitoring (MRM) verification also showed similar trends to the TMT results. In summary, our study is the first to investigate the adaptation of T. asahii under pressure from antifungal chemotherapy and host immune responses.

Genomic insights into the host specific adaptation of the Pneumocystis genus

Pneumocystis jirovecii, the fungal agent of human Pneumocystis pneumonia, is closely related to macaque Pneumocystis. Little is known about other Pneumocystis species in distantly related mammals, none of which are capable of establishing infection in humans. The molecular basis of host specificity in Pneumocystis remains unknown as experiments are limited due to an inability to culture any species in vitro. To explore Pneumocystis evolutionary adaptations, we have sequenced the genomes of species infecting macaques, rabbits, dogs and rats and compared them to available genomes of species infecting humans, mice and rats. Complete whole genome sequence data enables analysis and robust phylogeny, identification of important genetic features of the host adaptation, and estimation of speciation timing relative to the rise of their mammalian hosts. Our data reveals insights into the evolution of P. jirovecii, the sole member of the genus able to infect humans.

Principles of dengue virus evolvability derived from genotype-fitness maps in human and mosquito cells.

Dengue virus (DENV) cycles between mosquito and mammalian hosts. To examine how DENV populations adapt to these different host environments, we used serial passage in human and mosquito cell lines and estimated fitness effects for all single-nucleotide variants in these populations using ultra-deep sequencing. This allowed us to determine the contributions of beneficial and deleterious mutations to the collective fitness of the population. Our analysis revealed that the continuous influx of a large burden of deleterious mutations counterbalances the effect of rare, host-specific beneficial mutations to shape the path of adaptation. Beneficial mutations preferentially map to intrinsically disordered domains in the viral proteome and cluster to defined regions in the genome. These phenotypically redundant adaptive alleles may facilitate host-specific DENV adaptation. Importantly, the evolutionary constraints described in our simple system mirror trends observed across DENV and Zika strains, indicating it recapitulates key biophysical and biological constraints shaping long-term viral evolution.

Genetic Diversity and Phylogeography of Thottapalayam thottimvirus (Hantaviridae) in Asian House Shrew (Suncus murinus) in Eurasia.

Murid and cricetid rodents were previously believed to be the principal reservoir hosts of hantaviruses. Recently, however, multiple newfound hantaviruses have been discovered in shrews, moles, and bats, suggesting a complex evolutionary history. Little is known about the genetic diversity and geographic distribution of the prototype shrew-borne hantavirus, Thottapalayam thottimvirus (TPMV), carried by the Asian house shrew (Suncus murinus), which is widespread in Asia, Africa, and the Middle East. Comparison of TPMV genomic sequences from two Asian house shrews captured in Myanmar and Pakistan with TPMV strains in GenBank revealed that the Myanmar TPMV strain (H2763) was closely related to the prototype TPMV strain (VRC66412) from India. In the L-segment tree, on the other hand, the Pakistan TPMV strain (PK3629) appeared to be the most divergent, followed by TPMV strains from Nepal, then the Indian-Myanmar strains, and finally TPMV strains from China. The Myanmar strain of TPMV showed sequence similarity of 79.3–96.1% at the nucleotide level, but the deduced amino acid sequences showed a high degree of conservation of more than 94% with TPMV strains from Nepal, India, Pakistan, and China. Cophylogenetic analysis of host cytochrome b and TPMV strains suggested that the Pakistan TPMV strain was mismatched. Phylogenetic trees, based on host cytochrome b and cytochrome c oxidase subunit I genes of mitochondrial DNA, and on host recombination activating gene 1 of nuclear DNA, suggested that the Asian house shrew and Asian highland shrew (Suncus montanus) comprised a species complex. Overall, the geographic-specific clustering of TPMV strains in Asian countries suggested local host-specific adaptation. Additional in-depth studies are warranted to ascertain if TPMV originated in Asian house shrews on the Indian subcontinent.

Proteomic Analysis of Salmonella-modified Membranes Reveals Adaptations to Macrophage Hosts

Systemic infection and proliferation of intracellular pathogens require the biogenesis of a growth-stimulating compartment. The gastrointestinal pathogen Salmonella enterica commonly forms highly dynamic and extensive tubular membrane compartments built from Salmonella-modified membranes (SMMs) in diverse host cells. Although the general mechanism involved in the formation of replication-permissive compartments of S. enterica is well researched, much less is known regarding specific adaptations to different host cell types. Using an affinity-based proteome approach, we explored the composition of SMMs in murine macrophages. The systematic characterization provides a broader landscape of host players to the maturation of Salmonella-containing compartments and reveals core host elements targeted by Salmonella in macrophages as well as epithelial cells. However, we also identified subtle host specific adaptations. Some of these observations, such as the differential involvement of the COPII system, Rab GTPases 2A, 8B, 11 and ER transport proteins Sec61 and Sec22B may explain cell line-dependent variations in the pathophysiology of Salmonella infections. In summary, our system-wide approach demonstrates a hitherto underappreciated impact of the host cell type in the formation of intracellular compartments by Salmonella.