TADA: taxonomy-aware dataset aggregator.

SUMMARY: The profusion of sequenced genomes across the bacterial and archeal domains offers unprecedented possibilities for phylogenetic and comparative genomic analyses. In general, phylogenetic reconstruction is improved by the use of more data. However, including all available data is (i) not computationally tractable, and (ii) prone to biases, as the abundance of genomes is very unequally distributed over the biological diversity. Thus, in most cases, subsampling taxa to build a phylogeny is necessary. Currently, though, there is no available software to perform that handily. Here we present TADA, a taxonomic-aware dataset selection workflow that allows sampling across user-defined portions of the prokaryotic diversity with variable granularity, while setting constraints on genome quality and balance between branches. AVAILABILITY AND IMPLEMENTATION: TADA is implemented as a snakemake workflow and is freely available at https://github.com/emilhaegglund/TADA.

Deep mitochondrial origin outside the sampled alphaproteobacteria.

Mitochondria are ATP-generating organelles, the endosymbiotic origin of which was a key event in the evolution of eukaryotic cells 1 . Despite strong phylogenetic evidence that mitochondria had an alphaproteobacterial ancestry 2 , efforts to pinpoint their closest relatives among sampled alphaproteobacteria have generated conflicting results, complicating detailed inferences about the identity and nature of the mitochondrial ancestor. While most studies support the idea that mitochondria evolved from an ancestor related to Rickettsiales3-9, an order that includes several host-associated pathogenic and endosymbiotic lineages10,11, others have suggested that mitochondria evolved from a free-living group12-14. Here we re-evaluate the phylogenetic placement of mitochondria. We used genome-resolved binning of oceanic metagenome datasets and increased the genomic sampling of Alphaproteobacteria with twelve divergent clades, and one clade representing a sister group to all Alphaproteobacteria. Subsequent phylogenomic analyses that specifically address long branch attraction and compositional bias artefacts suggest that mitochondria did not evolve from Rickettsiales or any other currently recognized alphaproteobacterial lineage. Rather, our analyses indicate that mitochondria evolved from a proteobacterial lineage that branched off before the divergence of all sampled alphaproteobacteria. In light of this new result, previous hypotheses on the nature of the mitochondrial ancestor6,15,16 should be re-evaluated.

Host Adaptation in Legionellales Is 1.9 Ga, Coincident with Eukaryogenesis.

Bacteria adapting to living in a host cell caused the most salient events in the evolution of eukaryotes, namely the seminal fusion with an archaeon, and the emergence of both mitochondrion and chloroplast. A bacterial clade that may hold the key to understanding these events is the deep-branching gammaproteobacterial order Legionellales-containing among others Coxiella and Legionella-of which all known members grow inside eukaryotic cells. Here, by analyzing 35 novel Legionellales genomes mainly acquired through metagenomics, we show that this group is much more diverse than previously thought, and that key host-adaptation events took place very early in its evolution. Crucial virulence factors like the Type IVB secretion (Dot/Icm) system and two shared effector proteins were gained in the last Legionellales common ancestor (LLCA). Many metabolic gene families were lost in LLCA and its immediate descendants, including functions directly and indirectly related to molybdenum metabolism. On the other hand, genome sizes increased in the ancestors of the Legionella genus. We estimate that LLCA lived approximately 1.89 Ga, probably predating the last eukaryotic common ancestor by approximately 0.4-1.0 Gy. These elements strongly indicate that host adaptation arose only once in Legionellales, and that these bacteria were using advanced molecular machinery to exploit and manipulate host cells early in eukaryogenesis.

The evolution of intramitochondriality in Midichloria bacteria.

Midichloria spp. are intracellular bacterial symbionts of ticks. Representatives of this genus colonise mitochondria in the cells of their hosts. To shed light on this unique interaction we evaluated the presence of an intramitochondrial localization for three Midichloria in the respective tick host species and generated eight high-quality draft genomes and one closed genome, showing that this trait is non-monophyletic, either due to losses or multiple acquisitions. Comparative genomics supports the first hypothesis, as the genomes of non-mitochondrial symbionts are reduced subsets of those capable of colonising the organelles. We detect genomic signatures of mitochondrial tropism, including the differential presence of type IV secretion system and flagellum, which could allow the secretion of unique effectors and/or direct interaction with mitochondria. Other genes, including adhesion molecules, proteins involved in actin polymerisation, cell wall and outer membrane proteins, are only present in mitochondrial symbionts. The bacteria could use these to manipulate host structures, including mitochondrial membranes, to fuse with the organelles or manipulate the mitochondrial network.

Paralogization and New Protein Architectures in Planctomycetes Bacteria with Complex Cell Structures.

Bacteria of the phylum Planctomycetes have a unique cell plan with an elaborate intracellular membrane system, thereby resembling eukaryotic cells. The origin and evolution of these remarkable features is debated. To study the evolutionary genomics of bacteria with complex cell architectures, we have resequenced the 9.2-Mb genome of the model organism Gemmata obscuriglobus and sequenced the 10-Mb genome of G. massiliana Soil9, the 7.9-Mb genome of CJuql4, and the 6.7-Mb genome of Tuwongella immobilis, all of which belong to the family Gemmataceae. A gene flux analysis of the Planctomycetes revealed a massive emergence of novel protein families at multiple nodes within the Gemmataceae. The expanded protein families have unique multidomain architectures composed of domains that are characteristic of prokaryotes, such as the sigma factor domain of extracytoplasmic sigma factors, and domains that have proliferated in eukaryotes, such as the WD40, leucine-rich repeat, tetratricopeptide repeat and Ser/Thr kinase domains. Proteins with identifiable domains in the Gemmataceae have longer lengths and linkers than proteins in most other bacteria, and the analyses suggest that these traits were ancestrally present in the Planctomycetales. A broad comparison of protein length distribution profiles revealed an overlap between the longest proteins in prokaryotes and the shortest proteins in eukaryotes. We conclude that the many similarities between proteins in the Planctomycetales and the eukaryotes are due to convergent evolution and that there is no strict boundary between prokaryotes and eukaryotes with regard to features such as gene paralogy, protein length, and protein domain composition patterns.

Expression of the qepA1 gene is induced under antibiotic exposure.

BACKGROUND: The qepA1 gene encodes an efflux pump that reduces susceptibility to ciprofloxacin. Little is known about the regulation of qepA1 expression. OBJECTIVES: To assess the potential role of ciprofloxacin and other antibiotics in the regulation of qepA1 gene expression. To identify the promoter that drives qepA1 expression and other factors involved in expression regulation. To assess whether the identified features are universal among qepA alleles. METHODS: A translational qepA1-yfp fusion under the control of the qepA1 upstream region was cloned into the Escherichia coli chromosome. Expression of the fusion protein was measured in the presence of various antibiotics. Deletions within the upstream region were introduced to identify regions involved in gene expression and regulation. The qepA1 coding sequence and upstream region were compared with all available qepA sequences. RESULTS: Cellular stress caused by the presence of various antibiotics can induce qepA1 expression. The qepA1 gene is fused to a class I integron and gene expression is driven by the Pc promoter within the integrase gene. A segment within the integron belonging to a truncated dfrB4 gene is essential for the regulation of qepA1 expression. This genetic context is universal among all sequenced qepA alleles. CONCLUSIONS: The fusion of the qepA1 gene to a class I integron has created a novel regulatory unit that enables qepA1 expression to be under the control of antibiotic exposure. This setup mitigates potential negative effects of QepA1 production on bacterial fitness by restricting high-level expression to environmental conditions in which QepA1 is beneficial.

miComplete: weighted quality evaluation of assembled microbial genomes.

SUMMARY: Metagenomics and single-cell genomics have revolutionized the study of microorganisms, increasing our knowledge of microbial genomic diversity by orders of magnitude. A major issue pertaining to metagenome-assembled genomes (MAGs) and single-cell amplified genomes (SAGs) is to estimate their completeness and redundancy. Most approaches rely on counting conserved gene markers. In miComplete, we introduce a weighting strategy, where we normalize the presence/absence of markers by their median distance to the next marker in a set of complete reference genomes. This approach alleviates biases introduced by the presence/absence of shorter DNA pieces containing many markers, e.g. ribosomal protein operons. AVAILABILITY AND IMPLEMENTATION: miComplete is written in Python 3 and released under GPLv3. Source code and documentation are available at https://bitbucket.org/evolegiolab/micomplete. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Complex archaea that bridge the gap between prokaryotes and eukaryotes.

The origin of the eukaryotic cell remains one of the most contentious puzzles in modern biology. Recent studies have provided support for the emergence of the eukaryotic host cell from within the archaeal domain of life, but the identity and nature of the putative archaeal ancestor remain a subject of debate. Here we describe the discovery of 'Lokiarchaeota', a novel candidate archaeal phylum, which forms a monophyletic group with eukaryotes in phylogenomic analyses, and whose genomes encode an expanded repertoire of eukaryotic signature proteins that are suggestive of sophisticated membrane remodelling capabilities. Our results provide strong support for hypotheses in which the eukaryotic host evolved from a bona fide archaeon, and demonstrate that many components that underpin eukaryote-specific features were already present in that ancestor. This provided the host with a rich genomic 'starter-kit' to support the increase in the cellular and genomic complexity that is characteristic of eukaryotes.

Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA.

The distribution of fitness effects of mutations is a factor of fundamental importance in evolutionary biology. We determined the distribution of fitness effects of 510 mutants that each carried between 1 and 10 mutations (synonymous and nonsynonymous) in the hisA gene, encoding an essential enzyme in the l-histidine biosynthesis pathway of Salmonella enterica. For the full set of mutants, the distribution was bimodal with many apparently neutral mutations and many lethal mutations. For a subset of 81 single, nonsynonymous mutants most mutations appeared neutral at high expression levels, whereas at low expression levels only a few mutations were neutral. Furthermore, we examined how the magnitude of the observed fitness effects was correlated to several measures of biophysical properties and phylogenetic conservation.We conclude that for HisA: (i) The effect of mutations can be masked by high expression levels, such that mutations that are deleterious to the function of the protein can still be neutral with regard to organism fitness if the protein is expressed at a sufficiently high level; (ii) the shape of the fitness distribution is dependent on the extent to which the protein is rate-limiting for growth; (iii) negative epistatic interactions, on an average, amplified the combined effect of nonsynonymous mutations; and (iv) no single sequence-based predictor could confidently predict the fitness effects of mutations in HisA, but a combination of multiple predictors could predict the effect with a SD of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients.

Origin and Evolution of the Bartonella Gene Transfer Agent.

Gene transfer agents (GTAs) are domesticated bacteriophages that have evolved into molecular machines for the transfer of bacterial DNA. Despite their widespread nature and their biological implications, the mechanisms and selective forces that drive the emergence of GTAs are still poorly understood. Two GTAs have been identified in the Alphaproteobacteria: the RcGTA, which is widely distributed in a broad range of species; and the BaGTA, which has a restricted host range that includes vector-borne intracellular bacteria of the genus Bartonella. The RcGTA packages chromosomal DNA randomly, whereas the BaGTA particles contain a relatively higher fraction of genes for host interaction factors that are amplified from a nearby phage-derived origin of replication. In this study, we compare the BaGTA genes with homologous bacteriophage genes identified in the genomes of Bartonella species and close relatives. Unlike the BaGTA, the prophage genes are neither present in all species, nor inserted into homologous genomic sites. Phylogenetic inferences and substitution frequency analyses confirm codivergence of the BaGTA with the host genome, as opposed to multiple integration and recombination events in the prophages. Furthermore, the organization of segments flanking the BaGTA differs from that of the prophages by a few rearrangement events, which have abolished the normal coordination between phage genome replication and phage gene expression. Based on the results of our comparative analysis, we propose a model for how a prophage may be transformed into a GTA that transfers amplified bacterial DNA segments.

phyloSkeleton: taxon selection, data retrieval and marker identification for phylogenomics.

Summary: With the wealth of available genome sequences, a difficult and tedious part of inferring phylogenomic trees is now to select genomes with an appropriate taxon density in the different parts of the tree. The package described here offers tools to easily select the most representative organisms, following a set of simple rules based on taxonomy and assembly quality, to retrieve the genomes from public databases (NCBI, JGI), to annotate them if necessary, to identify given markers in these, and to prepare files for multiple sequence alignment. Availability and Implementation: phyloSkeleton is a Perl module and is freely available under GPLv3 at https://bitbucket.org/lionelguy/phyloskeleton/ . Contact: lionel.guy@imbim.uu.se.

Legionella pneumophila recurrently isolated in a Spanish hospital: Two years of antimicrobial resistance surveillance.

OBJECTIVES: The aim of this study was to monitor the spread, persistence and antibiotic resistance patterns of Legionella spp. strains found in a hospital water distribution system. These environmental studies are intended to help detect the presence of antibiotic resistant strains before they infect patients. METHODS: Antimicrobial surveillance tests were performed at 27 different sampling points of the water network of a large Spanish hospital over two years. Water samples were screened for Legionella according to ISO 11731:2007. Legionella spp. isolates were identified by serotyping and by mass spectrometry (MALDI-ToF). Epidemiological molecular typing was done by Pulse-Field Gel Electrophoresis (PFGE) and by Sequence-Based Typing (SBT). Antibiotic susceptibility tests were performed using disk diffusion and ETEST®. RESULTS: Legionella spp. were recurrently isolated for 2 years. All isolates belonged the same group, L. pneumophila serogroups 2-14. Isolates were all attributed by SBT to sequence type (ST) ST328, although PFGE revealed 5 different patterns. No significant change in antibiotic susceptibility could be observed for this study period, irrespectively of the method used. CONCLUSION: Colonization of water systems by Legionella spp. is still occurring, although all the prevention rules were strictly followed. Antibiotic resistance monitoring may help us to find resistance in bacteria with environmental reservoirs but difficult to isolate from patients. The knowledge of the antibiotic susceptibility in environmental strains may help us to predict changes in clinical strains. This study might also help reconsidering Legionnaires' disease (LD) diagnostic methods. L. pneumophila serogroups 2-14 present all along the time of the investigation in the water distribution system can cause LD. However, they may not be detected by routine urine tests run on patients, thereby missing an ongoing LD infection.

Resurrecting ancestral genes in bacteria to interpret ancient biosignatures.

Two datasets, the geologic record and the genetic content of extant organisms, provide complementary insights into the history of how key molecular components have shaped or driven global environmental and macroevolutionary trends. Changes in global physico-chemical modes over time are thought to be a consistent feature of this relationship between Earth and life, as life is thought to have been optimizing protein functions for the entirety of its approximately 3.8 billion years of history on the Earth. Organismal survival depends on how well critical genetic and metabolic components can adapt to their environments, reflecting an ability to optimize efficiently to changing conditions. The geologic record provides an array of biologically independent indicators of macroscale atmospheric and oceanic composition, but provides little in the way of the exact behaviour of the molecular components that influenced the compositions of these reservoirs. By reconstructing sequences of proteins that might have been present in ancient organisms, we can downselect to a subset of possible sequences that may have been optimized to these ancient environmental conditions. How can one use modern life to reconstruct ancestral behaviours? Configurations of ancient sequences can be inferred from the diversity of extant sequences, and then resurrected in the laboratory to ascertain their biochemical attributes. One way to augment sequence-based, single-gene methods to obtain a richer and more reliable picture of the deep past, is to resurrect inferred ancestral protein sequences in living organisms, where their phenotypes can be exposed in a complex molecular-systems context, and then to link consequences of those phenotypes to biosignatures that were preserved in the independent historical repository of the geological record. As a first step beyond single-molecule reconstruction to the study of functional molecular systems, we present here the ancestral sequence reconstruction of the beta-carbonic anhydrase protein. We assess how carbonic anhydrase proteins meet our selection criteria for reconstructing ancient biosignatures in the laboratory, which we term palaeophenotype reconstruction.This article is part of the themed issue 'Reconceptualizing the origins of life'.

Coetzeea brasiliensis gen. nov., sp. nov. isolated from larvae of Anopheles darlingi.

A Gram-stain-negative, rod-shaped strain, Braz8T, isolated from larvae of Anopheles darlingi was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Braz8T was related most closely to species of the genus Thorsellia, with 95.6, 96.5 and 96.6 % similarity to the type strains of Thorsellia anophelis, Thorsellia kandunguensis and Thorsellia kenyensis, respectively, and formed a separate branch in the phylogenetic tree next to the monophyletic cluster of the genus Thorsellia. Chemotaxonomic data supported the allocation of the strain to the family Thorselliaceae. The major fatty acids were C18 : 1ω7c, C16 : 0 and C14 : 0. The quinone system was composed of ubiquinones Q-8 and Q-7 (1 : 0.3), the predominant polar lipids were diphosphatidylglycerol and phosphatidylglycerol, and the polyamine pattern showed the major compound putrescine. However, qualitative and quantitative differences in the major polyamine, polar lipid profile and fatty acid patterns distinguished strain Braz8T from species of the genus Thorsellia. Phylogenetic analysis based on 16S rRNA gene sequences, average nucleotide identity, DNA-DNA hybridization, multilocus sequence analysis as well as physiological and biochemical tests distinguished strain Braz8T both genotypically and phenotypically from the three Thorsellia species but also showed its placement in the family Thorselliaceae. Thus, strain Braz8T is considered to represent a novel species of a new genus most closely related to the genus Thorsellia, for which the name Coetzeea brasiliensis gen. nov., sp. nov. is proposed. The type strain of Coetzeea brasiliensis is Braz8T (=LMG 29552T=CIP 111088T).

A gene transfer agent and a dynamic repertoire of secretion systems hold the keys to the explosive radiation of the emerging pathogen Bartonella.

Gene transfer agents (GTAs) randomly transfer short fragments of a bacterial genome. A novel putative GTA was recently discovered in the mouse-infecting bacterium Bartonella grahamii. Although GTAs are widespread in phylogenetically diverse bacteria, their role in evolution is largely unknown. Here, we present a comparative analysis of 16 Bartonella genomes ranging from 1.4 to 2.6 Mb in size, including six novel genomes from Bartonella isolated from a cow, two moose, two dogs, and a kangaroo. A phylogenetic tree inferred from 428 orthologous core genes indicates that the deadly human pathogen B. bacilliformis is related to the ruminant-adapted clade, rather than being the earliest diverging species in the genus as previously thought. A gene flux analysis identified 12 genes for a GTA and a phage-derived origin of replication as the most conserved innovations. These are located in a region of a few hundred kb that also contains 8 insertions of gene clusters for type III, IV, and V secretion systems, and genes for putatively secreted molecules such as cholera-like toxins. The phylogenies indicate a recent transfer of seven genes in the virB gene cluster for a type IV secretion system from a cat-adapted B. henselae to a dog-adapted B. vinsonii strain. We show that the B. henselae GTA is functional and can transfer genes in vitro. We suggest that the maintenance of the GTA is driven by selection to increase the likelihood of horizontal gene transfer and argue that this process is beneficial at the population level, by facilitating adaptive evolution of the host-adaptation systems and thereby expansion of the host range size. The process counters gene loss and forces all cells to contribute to the production of the GTA and the secreted molecules. The results advance our understanding of the role that GTAs play for the evolution of bacterial genomes.

Proposal of Thorsellia kenyensis sp. nov. and Thorsellia kandunguensis sp. nov., isolated from larvae of Anopheles arabiensis, as members of the family Thorselliaceae fam. nov.

Two Gram-negative, rod-shaped strains, T2.1(T) and W5.1.1(T), isolated from larvae of the mosquito Anopheles arabiensis, were investigated using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity studies, strains T2.1(T) and W5.1.1(T) were shown to belong to the genus Thorsellia, both showing 97.8 % similarity to the type strain of Thorsellia anophelis, with 98.1 % similarity to each other. Chemotaxonomic data supported the allocation of the strains to the genus Thorsellia: their major fatty acids were C18 : 1ω7c, C16 : 0 and C14 : 0 and they harboured a ubiquinone Q-8 quinone system and a polyamine pattern with the major compound 1,3-diaminopropane. Qualitative and quantitative differences in their polar lipid profiles distinguished strains T2.1(T) and W5.1.1(T) from each other and from T. anophelis. Average nucleotide identity (ANI), DNA-DNA hybridization, multilocus sequence analysis (MLSA) as well as physiological and biochemical tests allowed T2.1(T) and W5.1.1(T) to be distinguished both genotypically and phenotypically from each other and from the type strain of T. anophelis. Thus, we propose that these isolates represent two novel species of the genus Thorsellia, named Thorsellia kenyensis sp. nov. (type strain T2.1(T) = CCM 8545(T) = LMG 28483(T) = CIP 110829(T)) and Thorsellia kandunguensis sp. nov. (type strain W5.1.1(T) = LMG 28213(T) = CIP 110794(T)). Furthermore, phylogenetic analysis based on nearly full-length 16S rRNA gene sequences showed that the genus Thorsellia forms a separate branch, distinct from the families Enterobacteriaceae, Pasteurellaceae and Orbaceae. As a consequence, a new family Thorselliaceae fam. nov. is proposed. An emended description of Thorsellia anophelis is also provided.

Phylogeny and Expansion of Serine/Threonine Kinases in Phagocytotic Bacteria in the Phylum Planctomycetota.

The recently isolated bacterium "Candidatus Uabimicrobium amorphum" is the only known prokaryote that can engulf other bacterial cells. Its proteome contains a high fraction of proteins involved in signal transduction systems, which is a feature normally associated with multicellularity in eukaryotes. Here, we present a protein-based phylogeny which shows that "Ca. Uabimicrobium amorphum" represents an early diverging lineage that clusters with the Saltatorellus clade within the phylum Planctomycetota. A gene flux analysis indicated a gain of 126 protein families for signal transduction functions in "Ca. Uabimicrobium amorphum", of which 66 families contained eukaryotic-like Serine/Threonine kinases with Pkinase domains. In total, we predicted 525 functional Serine/Threonine kinases in "Ca. Uabimicrobium amorphum", which represent 8% of the proteome and is the highest fraction of Serine/Threonine kinases in a bacterial proteome. The majority of Serine/Threonine kinases in this species are membrane proteins and 30% contain long, tandem arrays of WD40 or TPR domains. The pKinase domain was predicted to be located in the cytoplasm, while the WD40 and TPR domains were predicted to be located in the periplasm. Such domain combinations were also identified in the Serine/Threonine kinases of other species in the Planctomycetota, although in much lower abundances. A phylogenetic analysis of the Serine/Threonine kinases in the Planctomycetota inferred from the Pkinase domain alone provided support for lineage-specific expansions of the Serine/Threonine kinases in "Ca. Uabimicrobium amorphum". The results imply that expansions of eukaryotic-like signal transduction systems are not restricted to multicellular organisms, but have occurred in parallel in prokaryotes with predatory lifestyles and phagocytotic-like behaviors.

Close encounters of the third domain: the emerging genomic view of archaeal diversity and evolution.

The Archaea represent the so-called Third Domain of life, which has evolved in parallel with the Bacteria and which is implicated to have played a pivotal role in the emergence of the eukaryotic domain of life. Recent progress in genomic sequencing technologies and cultivation-independent methods has started to unearth a plethora of data of novel, uncultivated archaeal lineages. Here, we review how the availability of such genomic data has revealed several important insights into the diversity, ecological relevance, metabolic capacity, and the origin and evolution of the archaeal domain of life.

Rapid adaptations of Legionella pneumophila to the human host.

Legionella pneumophila are host-adapted bacteria that infect and reproduce primarily in amoeboid protists. Using similar infection mechanisms, they infect human macrophages, and cause Legionnaires' disease, an atypical pneumonia, and the milder Pontiac fever. We hypothesized that, despite the similarities in infection mechanisms, the hosts are different enough that there exist high-selective value mutations that would dramatically increase the fitness of Legionella inside the human host. By comparing a large number of isolates from independent infections, we identified two genes, mutated in three unrelated patients, despite the short duration of the incubation period (2-14 days). One is a gene coding for an outer membrane protein (OMP) belonging to the OmpP1/FadL family. The other is a gene coding for an EAL-domain-containing protein involved in cyclic-di-GMP regulation, which in turn modulates flagellar activity. The clinical strain, carrying the mutated EAL-domain-containing homologue, grows faster in macrophages than the wild-type strain, and thus appears to be better adapted to the human host. As human-to-human transmission is very rare, fixation of these mutations into the population and spread into the environment is unlikely. Therefore, parallel evolution - here mutations in the same genes observed in independent human infections - could point to adaptations to the accidental human host. These results suggest that despite the ability of L. pneumophila to infect, replicate in and exit from macrophages, its human-specific adaptations are unlikely to be fixed in the population.