Minghsun Liu

Propionibacterium acnes Strain Populations in the Human Skin Microbiome Associated with Acne

The human skin microbiome plays important roles in skin health and disease. However, bacterial population structure and diversity at the strain level is poorly understood. We compared the skin microbiome at the strain level and genome level of Propionibacterium acnes, a dominant skin commensal, between 49 acne patients and 52 healthy individuals by sampling the pilosebaceous units on their noses. Metagenomic analysis demonstrated that while the relative abundances of P. acnes were similar, the strain population structures were significantly different in the two cohorts. Certain strains were highly associated with acne and other strains were enriched in healthy skin. By sequencing 66 previously unreported P. acnes strains and comparing 71 P. acnes genomes, we identified potential genetic determinants of various P. acnes strains in association with acne or health. Our analysis suggests that acquired DNA sequences and bacterial immune elements may play roles in determining virulence properties of P. acnes strains and some could be future targets for therapeutic interventions. This study demonstrates a previously unreported paradigm of commensal strain populations that could explain the pathogenesis of human diseases. It underscores the importance of strain level analysis of the human microbiome to define the role of commensals in health and disease.

Original ArticlePropionibacterium acnes Strain Populations in the Human Skin Microbiome Associated with Acne

The human skin microbiome plays important roles in skin health and disease. However, bacterial population structure and diversity at the strain level is poorly understood. We compared the skin microbiome at the strain level and genome level of Propionibacterium acnes, a dominant skin commensal, between 49 acne patients and 52 healthy individuals by sampling the pilosebaceous units on their noses. Metagenomic analysis demonstrated that while the relative abundances of P. acnes were similar, the strain population structures were significantly different in the two cohorts. Certain strains were highly associated with acne and other strains were enriched in healthy skin. By sequencing 66 previously unreported P. acnes strains and comparing 71 P. acnes genomes, we identified potential genetic determinants of various P. acnes strains in association with acne or health. Our analysis suggests that acquired DNA sequences and bacterial immune elements may play roles in determining virulence properties of P. acnes strains and some could be future targets for therapeutic interventions. This study demonstrates a previously unreported paradigm of commensal strain populations that could explain the pathogenesis of human diseases. It underscores the importance of strain level analysis of the human microbiome to define the role of commensals in health and disease.

Tropism switching in Bordetella bacteriophage defines a family of diversity-generating retroelements

Bordetella bacteriophages generate diversity in a gene that specifies host tropism. This microevolutionary adaptation is produced by a genetic element that combines the basic retroelement life cycle of transcription, reverse transcription and integration with site-directed, adenine-specific mutagenesis. Central to this process is a reverse transcriptase-mediated exchange between two repeats; one serving as a donor template (TR) and the other as a recipient of variable sequence information (VR). Here we describe the genetic basis for diversity generation. The directionality of information transfer is determined by a 21-base-pair sequence present at the 3′ end of VR. On the basis of patterns of marker transfer in response to variant selective pressures, we propose that a TR reverse transcript is mutagenized, integrated into VR as a single non-coding strand, and then partially converted to the parental VR sequence. This allows the diversity-generating system to minimize variability to the subset of bases under selection. Using the Bordetella phage cassette as a signature, we have identified numerous related elements in diverse bacteria. These elements constitute a new family of retroelements with the potential to confer selective advantages to their host genomes.

Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade

Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SSBsa) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SSBsa activity. Although intracellular movement was essential for cell–cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell–cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SSBsa-mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.

Comparative genomics of the classical Bordetella subspecies: the evolution and exchange of virulence-associated diversity amongst closely related pathogens

BackgroundThe classical Bordetella subspecies are phylogenetically closely related, yet differ in some of the most interesting and important characteristics of pathogens, such as host range, virulence and persistence. The compelling picture from previous comparisons of the three sequenced genomes was of genome degradation, with substantial loss of genome content (up to 24%) associated with adaptation to humans.ResultsFor a more comprehensive picture of lineage evolution, we employed comparative genomic and phylogenomic analyses using seven additional diverse, newly sequenced Bordetella isolates. Genome-wide single nucleotide polymorphism (SNP) analysis supports a reevaluation of the phylogenetic relationships between the classical Bordetella subspecies, and suggests a closer link between ovine and human B. parapertussis lineages than has been previously proposed. Comparative analyses of genome content revealed that only 50% of the pan-genome is conserved in all strains, reflecting substantial diversity of genome content in these closely related pathogens that may relate to their different host ranges, virulence and persistence characteristics. Strikingly, these analyses suggest possible horizontal gene transfer (HGT) events in multiple loci encoding virulence factors, including O-antigen and pertussis toxin (Ptx). Segments of the pertussis toxin locus (ptx) and its secretion system locus (ptl) appear to have been acquired by the classical Bordetella subspecies and are divergent in different lineages, suggesting functional divergence in the classical Bordetellae.ConclusionsTogether, these observations, especially in key virulence factors, reveal that multiple mechanisms, such as point mutations, gain or loss of genes, as well as HGTs, contribute to the substantial phenotypic diversity of these versatile subspecies in various hosts.

Genomic and Genetic Analysis of Bordetella Bacteriophages Encoding Reverse Transcriptase-Mediated Tropism-Switching Cassettes

Liu et al. recently described a group of related temperate bacteriophages that infect Bordetella subspecies and undergo a unique template-dependent, reverse transcriptase-mediated tropism switching phenomenon (Liu et al., Science 295: 2091-2094, 2002). Tropism switching results from the introduction of single nucleotide substitutions at defined locations in the VR1 (variable region 1) segment of the mtd (major tropism determinant) gene, which determines specificity for receptors on host bacteria. In this report, we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability. Forty-nine coding sequences were identified. Of these coding sequences, bbp36 contained VR2 (variable region 2), which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and bpm encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations. Morphological and sequence analysis suggests that these Bordetella phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes. Bordetella bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take place in the mouse respiratory tract during infection.

Surface display of a massively variable lipoprotein by a Legionella diversity-generating retroelement

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by facilitating localized DNA sequence evolution through a specialized error-prone reverse transcription process. We characterized a DGR in Legionella pneumophila, an opportunistic human pathogen that causes Legionnaires disease. The L. pneumophila DGR is found within a horizontally acquired genomic island, and it can theoretically generate 1026 unique nucleotide sequences in its target gene, legionella determinent target A (ldtA), creating a repertoire of 1019 distinct proteins. Expression of the L. pneumophila DGR resulted in transfer of DNA sequence information from a template repeat to a variable repeat (VR) accompanied by adenine-specific mutagenesis of progeny VRs at the 3′end of ldtA. ldtA encodes a twin-arginine translocated lipoprotein that is anchored in the outer leaflet of the outer membrane, with its C-terminal variable region surface exposed. Related DGRs were identified in L. pneumophila clinical isolates that encode unique target proteins with homologous VRs, demonstrating the adaptability of DGR components. This work characterizes a DGR that diversifies a bacterial protein and confirms the hypothesis that DGR-mediated mutagenic homing occurs through a conserved mechanism. Comparative bioinformatics predicts that surface display of massively variable proteins is a defining feature of a subset of bacterial DGRs.

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin.

The viral population, including bacteriophages, is an important component of the human microbiota, yet is poorly understood. We aim to determine whether bacteriophages modulate the composition of the bacterial populations, thus potentially playing a role in health or disease. We investigated the diversity and host interactions of the bacteriophages of Propionibacterium acnes, a major human skin commensal implicated in acne pathogenesis. By sequencing 48 P. acnes phages isolated from acne patients and healthy individuals and by analyzing the P. acnes phage populations in healthy skin metagenomes, we revealed that P. acnes phage populations in the skin microbial community are often dominated by one strain. We also found phage strains shared among both related and unrelated individuals, suggesting that a pool of common phages exists in the human population and that transmission of phages may occur between individuals. To better understand the bacterium–phage interactions in the skin microbiota, we determined the outcomes of 74 genetically defined Propionibacterium strains challenged by 15 sequenced phages. Depending on the Propionibacterium lineage, phage infection can result in lysis, pseudolysogeny, or resistance. In type II P. acnes strains, we found that encoding matching clustered regularly interspaced short palindromic repeat spacers is insufficient to confer phage resistance. Overall, our findings suggest that the prey–predator relationship between bacteria and phages may have a role in modulating the composition of the microbiota. Our study also suggests that the microbiome structure of an individual may be an important factor in the design of phage-based therapy.

Early days: genomics and human responses to infection

DNA microarray-based gene transcript-profiling of the responses of primates to infection has begun to yield new insights into host–pathogen interactions; this approach, however, remains plagued by challenges and complexities that have yet to be adequately addressed. The rapidly changing nature over time of acute infectious diseases in a host, and the genetic diversity of microbial pathogens present unique problems for the design and interpretation of functional-genomic studies in this field. In addition, there are the more common problems related to heterogeneity within clinical samples, the complex, non-standardized confounding variables associated with human subjects and the complexities posed by the analysis and validation of highly parallel data. Whereas various approaches have been developed to address each of these issues, there are significant limitations that remain to be overcome. The resolution of these problems should lead to a better understanding of the dialogue between the host and pathogen.

Transcriptome patterns from primary cutaneous Leishmania braziliensis infections associate with eventual development of mucosal disease in humans.

Introduction Localized Cutaneous Leishmaniasis (LCL) and Mucosal Leishmaniasis (ML) are two extreme clinical forms of American Tegumentary Leishmaniasis that usually begin as solitary primary cutaneous lesions. Host and parasite factors that influence the progression of LCL to ML are not completely understood. In this manuscript, we compare the gene expression profiles of primary cutaneous lesions from patients who eventually developed ML to those that did not. Methods Using RNA-seq, we analyzed both the human and Leishmania transcriptomes in primary cutaneous lesions. Results Limited number of reads mapping to Leishmania transcripts were obtained. For human transcripts, compared to ML patients, lesions from LCL patients displayed a general multi-polarization of the adaptive immune response and showed up-regulation of genes involved in chemoattraction of innate immune cells and in antigen presentation. We also identified a potential transcriptional signature in the primary lesions that may predict long-term disease outcome. Conclusions We were able to simultaneously sequence both human and Leishmania mRNA transcripts in primary cutaneous leishmaniasis lesions. Our results suggest an intrinsic difference in the immune capacity of LCL and ML patients. The findings correlate the complete cure of L. braziliensis infection with a controlled inflammatory response and a balanced activation of innate and adaptive immunity.