In many human infections, hosts and pathogens coexist for years or decades. Important examples include HIV, herpes viruses, tuberculosis, leprosy, and malaria. With the exception of intensively studied viral infections such as HIV͞AIDs, little is known about the extent to which the clonal expansion that occurs during long-term infection by pathogens involves important genetic adaptations. We report here a detailed, whole-genome analysis of one such infection, that of a cystic fibrosis (CF) patient by the opportunistic bacterial pathogen Pseudomonas aeruginosa. The bacteria underwent numerous genetic adaptations during 8 years of infection, as evidenced by a positive-selection signal across the genome and an overwhelming signal in specific genes, several of which are mutated during the course of most CF infections. Of particular interest is our finding that virulence factors that are required for the initiation of acute infections are often selected against during chronic infections. It is apparent that the genotypes of the P. aeruginosa strains present in advanced CF infections differ systematically from those of ''wild-type'' P. aeruginosa and that these differences may offer new opportunities for treatment of this chronic disease.chronic infection ͉ positive selection ͉ virulence ͉ antibiotic resistance M ost cystic fibrosis (CF) patients acquire chronic Pseudomonas aeruginosa infections by their teenage years, if not earlier, and these respiratory infections are responsible for much of the morbidity and mortality caused by CF (1, 2). It has been established that most of these infections are clonal (3), and even among groups of CF patients treated in specific clinics the infections are acquired independently, presumably from diverse environmental reservoirs (4). Previous studies, particularly of the O-antigen biosynthetic locus and the transcriptional regulator mucA, indicate that some P. aeruginosa genes commonly incur loss-of-function mutations as the infections progress (5-7). Mutator phenotypes also arise frequently (8).The overall picture is reminiscent of typical cancers: a clone of cells, albeit in this instance one of exogenous origin, experiences selection for an accumulation of genetic variants that promote long-term survival and clonal expansion. Our data validate this model for P. aeruginosa infections in CF and provide strong evidence for the role of selection in shaping the genotypes of the bacteria that are present during the late, life-threatening phase of the infections. Our data also focus attention on particular aspects of P. aeruginosa metabolism that are premier targets of selection, both in the patient we studied in most detail and in other, independently evolving, P. aeruginosa infections in additional CF patients.
SummaryThe opportunistic pathogen Pseudomonas aeruginosa undergoes genetic change during chronic airway infection of cystic fibrosis (CF) patients. One common change is a mutation inactivating lasR, which encodes a transcriptional regulator that responds to a homoserine lactone signal to activate expression of acute virulence factors. Colonies of lasR mutants visibly accumulated the iridescent intercellular signal 4-hydroxy-2-heptylquinoline. Using this colony phenotype, we identified P. aeruginosa lasR mutants that emerged in the airway of a CF patient early during chronic infection, and during growth in the laboratory on a rich medium. The lasR loss-of-function mutations in these strains conferred a growth advantage with particular carbon and nitrogen sources, including amino acids, in part due to increased expression of the catabolic pathway regulator CbrB. This growth phenotype could contribute to selection of lasR mutants both on rich medium and within the CF airway, supporting a key role for bacterial metabolic adaptation during chronic infection. Inactivation of lasR also resulted in increased b-lactamase activity that increased tolerance to ceftazidime, a widely used b-lactam antibiotic. Loss of LasR function may represent a marker of an early stage in chronic infection of the CF airway with clinical implications for antibiotic resistance and disease progression.
) it was shown that Pseudomonas aeruginosa undergoes intense genetic adaptation during chronic respiratory infection (CRI) in cystic fibrosis (CF) patients. We used the same collection of isolates to explore the role of hypermutation in this process, since one of the hallmarks of CRI is the high prevalence of DNA mismatch repair (MMR) system-deficient mutator strains. The presence of mutations in 34 genes (many of them positively linked to adaptation in CF patients) in the study collection of 90 P. aeruginosa isolates obtained longitudinally from 29 CF patients was not homogeneous; on the contrary, mutations were significantly concentrated in the mutator lineages, which represented 17% of the isolates (87% MMR deficient). While sequential nonmutator lineages acquired a median of only 0.25 mutation per year of infection, mutator lineages accumulated more than 3 mutations per year. On the whole-genome scale, data for the first fully sequenced late CF isolate, which was also shown to be an MMR-deficient mutator, also support these findings. Moreover, for the first time the predicted amplification of mutator populations due to hitchhiking with adaptive mutations in the course of natural human infections is clearly documented. Interestingly, increased accumulation of mutations in mutator lineages was not a consequence of overrepresentation of mutations in genes involved in antimicrobial resistance, the only adaptive trait linked so far to hypermutation in CF patients, demonstrating that hypermutation also plays a major role in P. aeruginosa genome evolution and adaptation during CRI.Chronic respiratory infection (CRI) with Pseudomonas aeruginosa is the main driver of morbidity and mortality in cystic fibrosis (CF) patients (4, 15). The establishment of P. aeruginosa CRI is mediated by a complex adaptive process that includes physiological changes produced by the activation of specific regulatory pathways, including the induction of the biofilm mode of growth or the differential expression of virulence genes (39), and genetic changes leading to selection of an important number of adaptive mutations required for long-term persistence (19,27,34).Although CF patients with CRI are generally infected by a single P. aeruginosa strain that in most cases persists throughout the patient's life (32), one of the hallmarks of such infections is the emergence and fixation over time of multiple phenotypic variants of the underlying clonal populations (24), a process known as adaptive radiation (30). Many of the selected phenotypes have been clearly linked to adaptation to the lung environment that favors lifelong persistence of CRI (27). Indeed, once the adaptation stage is reached and the CRI is fully established, eradication is generally no longer possible. At this point, with resignation, the therapeutic goals change from attempting to cure the infection to slowing the decline of lung function and improving the patient's quality of life.The intense genetic adaptation process that takes place during the establishment of CRI has r...
Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa. The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa. There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R, previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY, a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.
Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, ϳ10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core P. aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.
Apolipoprotein L1 (APOL1) is a human protein that confers immunity to Trypanosoma brucei infections but can be countered by a trypanosome-encoded antagonist SRA. APOL1 belongs to a family of programmed cell death genes whose proteins can initiate host apoptosis or autophagic death. We report here that all six members of the APOL gene family (APOL1-6) present in humans have rapidly evolved in simian primates. APOL6, furthermore, shows evidence of an adaptive sweep during recent human evolution. In each APOL gene tested, we found rapidly evolving codons in or adjacent to the SRA-interacting protein domain (SID), which is the domain of APOL1 that interacts with SRA. In APOL6, we also found a rapidly changing 13-amino-acid cluster in the membrane-addressing domain (MAD), which putatively functions as a pH sensor and regulator of cell death. We predict that APOL genes are antagonized by pathogens by at least two distinct mechanisms: SID antagonists, which include SRA, that interact with the SID of various APOL proteins, and MAD antagonists that interact with the MAD hinge base of APOL6. These antagonists either block or prematurely cause APOL-mediated programmed cell death of host cells to benefit the infecting pathogen. These putative interactions must occur inside host cells, in contrast to secreted APOL1 that trafficks to the trypanosome lysosome. Hence, the dynamic APOL gene family appears to be an important link between programmed cell death of host cells and immunity to pathogens.
COVID-19 patients often develop severe cardiovascular complications, but it remains unclear if these are caused directly by viral infection or are secondary to a systemic response. Here we examine the cardiac tropism of SARS-CoV-2 in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and smooth muscle cells (hPSC-SMCs). We find that that SARS-CoV-2 selectively infects hPSC-CMs through the viral receptor ACE2, whereas in hPSC-SMCs there is minimal viral entry or replication. After entry into cardiomyocytes, SARS-CoV-2 is assembled in lysosome-like vesicles and egresses via bulk exocytosis. The viral transcripts become a large fraction of cellular mRNA while host gene expression shifts from oxidative to glycolytic metabolism and up-regulates chromatin modification and RNA splicing pathways. Most importantly, viral infection of hPSC-CMs progressively impairs both their electrophysiological and contractile function, and causes widespread cell death. These data support the hypothesis that COVID-19-related cardiac symptoms can result from a direct cardiotoxic effect of SARS-CoV-2.
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