Bacterial classification using genomic fingerprints obtained by virtual hybridization

In silico genomic fingerprints were produced by virtual hybridization of 191 fully sequenced bacterial genomes using a set of 15,264 13-mer probes specially designed to produce universal whole genome fingerprints. A novel approach for constructing phylogenetic trees, based on comparative analysis of genomic fingerprints, was developed. The resultant bacterial phylogenetic tree had strong similarities to those produced from the alignment of conserved sequences. Notably, the trees derived from the alignment of other conserved COG genes divided the Bacillus and Corynebacterium genera into the same subgroups produced by the novel bacterial tree. A number of discrepancies between both techniques were observed for the grouping of some Lactobacillus species. However, a detailed analysis of the alignment of these genomes using other bioinformatics tools revealed that the grouping of these organisms in the novel tree was more satisfactory than the groupings from previous classifications, which used only a few conserved genes. All these data suggest that the bacterial taxonomy produced by genomic fingerprints is satisfactory, but sometimes different from classical taxonomies. Discrepancies probably arise because the fingerprinting technique analyzes genomic sequences and reveals more information than previously used approaches.     

Reciprocal adaptation of rice and Xanthomonas oryzae pv. oryzae: cross-species 2D GWAS reveals the underlying genetics

A 1D/2D genome-wide association study strategy was adopted to investigate the genetic systems underlying the reciprocal adaptation of rice (Oryza sativa) and its bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo) using the whole-genome sequencing and large-scale phenotyping data of 701 rice accessions and 23 diverse Xoo strains. Forty-seven Xoo virulence-related genes and 318 rice quantitative resistance genes (QR-genes) mainly located in 41 genomic regions, and genome-wide interactions between the detected virulence-related genes and QR genes were identified, including well-known resistance genes/virulence genes plus many previously uncharacterized ones. The relationship between rice and Xoo was characterized by strong differentiation among Xoo races corresponding to the subspecific differentiation of rice, by strong shifts toward increased resistance/virulence of rice/Xoo populations and by rich genetic diversity at the detected rice QR-genes and Xoo virulence genes, and by genome-wide interactions between many rice QR-genes and Xoo virulence genes in a multiple-to-multiple manner, presumably resulting either from direct protein–protein interactions or from genetic epistasis. The observed complex genetic interaction system between rice and Xoo likely exists in other crop–pathogen systems that would maintain high levels of diversity at their QR-loci/virulence-loci, resulting in dynamic coevolutionary consequences during their reciprocal adaptation.

A complex system of genetic interactions leads to reciprocal adaptation between rice and its bacterial pathogen, Xanthomonas oryzae pv. oryzae.     

Swimming Motility in a Longitudinal Collection of Clinical Isolates of Burkholderia cepacia Complex Bacteria from People with Cystic Fibrosis

Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.     

Bacterial classification using genomic fingerprints obtained by virtual hybridization

In silico genomic fingerprints were produced by virtual hybridization of 191 fully sequenced bacterial genomes using a set of 15,264 13-mer probes specially designed to produce universal whole genome fingerprints. A novel approach for constructing phylogenetic trees, based on comparative analysis of genomic fingerprints, was developed. The resultant bacterial phylogenetic tree had strong similarities to those produced from the alignment of conserved sequences. Notably, the trees derived from the alignment of other conserved COG genes divided the Bacillus and Corynebacterium genera into the same subgroups produced by the novel bacterial tree. A number of discrepancies between both techniques were observed for the grouping of some Lactobacillus species. However, a detailed analysis of the alignment of these genomes using other bioinformatics tools revealed that the grouping of these organisms in the novel tree was more satisfactory than the groupings from previous classifications, which used only a few conserved genes. All these data suggest that the bacterial taxonomy produced by genomic fingerprints is satisfactory, but sometimes different from classical taxonomies. Discrepancies probably arise because the fingerprinting technique analyzes genomic sequences and reveals more information than previously used approaches.     

Comparative Genomic Analysis Reveals a Possible Novel Non-Tuberculous Mycobacterium Species with High Pathogenic Potential

Mycobacteria have been reported to cause a wide range of human diseases. We present the first whole-genome study of a Non-Tuberculous Mycobacterium, Mycobacterium sp. UM_CSW (referred to hereafter as UM_CSW), isolated from a patient diagnosed with bronchiectasis. Our data suggest that this clinical isolate is likely a novel mycobacterial species, supported by clear evidence from molecular phylogenetic, comparative genomic, ANI and AAI analyses. UM_CSW is closely related to the Mycobacterium avium complex. While it has characteristic features of an environmental bacterium, it also shows a high pathogenic potential with the presence of a wide variety of putative genes related to bacterial virulence and shares very similar pathogenomic profiles with the known pathogenic mycobacterial species. Thus, we conclude that this possible novel Mycobacterium species should be tightly monitored for its possible causative role in human infections.     

Role of amylopectin synthesis in Toxoplasma gondii and its implication in vaccine development against toxoplasmosis

Toxoplasma gondii is a ubiquitous pathogen infecting one-third of the global population. A significant fraction of toxoplasmosis cases is caused by reactivation of existing chronic infections. The encysted bradyzoites during chronic infection accumulate high levels of amylopectin that is barely present in fast-replicating tachyzoites. However, the physiological significance of amylopectin is not fully understood. Here, we identified a starch synthase (SS) that is required for amylopectin synthesis in T. gondii. Genetic ablation of SS abolished amylopectin production, reduced tachyzoite proliferation, and impaired the recrudescence of bradyzoites to tachyzoites. Disruption of the parasite Ca²⁺-dependent protein kinase 2 (CDPK2) was previously shown to cause massive amylopectin accumulation and bradyzoite death. Therefore, the Δcdpk2 mutant is thought to be a vaccine candidate. Notably, deleting SS in a Δcdpk2 mutant completely abolished starch accrual and restored cyst formation as well as virulence in mice. Together these results suggest that regulated amylopectin production is critical for the optimal growth, development and virulence of Toxoplasma. Not least, our data underscore a potential drawback of the Δcdpk2 mutant as a vaccine candidate as it may regain full virulence by mutating amylopectin synthesis genes like SS.     

Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections.

Pathogenic microbes have evolved highly sophisticated mechanisms for colonizing host tissues and evading or deflecting assault by the immune response. The ability of these microbes to avoid clearance prolongs infection, thereby promoting their long-term survival within individual hosts and, through transmission, between hosts. Many pathogens are capable of extensive antigenic changes in the face of the multiple constitutive and dynamic components of host immune defenses. As a result, highly diverse populations that have widely different virulence properties can arise from a single infecting organism (clone). In this review, we consider the molecular and genetic features of antigenic variation and corresponding host-parasite interactions of different pathogenic bacterial, fungal, and protozoan microorganisms. The host and microbial molecules involved in these interactions often determine the adhesive, invasive, and antigenic properties of the infecting organisms and can dramatically affect the virulence and pathobiology of individual infections. Pathogens capable of such antigenic variation exhibit mechanisms of rapid mutability in confined chromosomal regions containing specialized genes designated contingency genes. The mechanisms of hypermutability of contingency genes are common to a variety of bacterial and eukaryotic pathogens and include promoter alterations, reading-frame shifts, gene conversion events, genomic rearrangements, and point mutations.     

Identification of secreted virulence factors of Chromobacterium violaceum

Chromobacterium violaceum, a component of tropical soil microbiota, is an opportunistic pathogenic bacterium that can infect humans and other animals. In addition to identifying a large number of genes that demonstrate the vast biotechnological potential of this bacterium, genome sequencing revealed several virulence factors, including different cytolysins, which can be related to its pathogenicity. Here we confirmed these predictions from genomic analyses by identifying, through mass spectrometry, proteins present in the culture supernatant of C. violaceum that may constitute secreted virulence factors. Among them, we identified a secreted collagenase and the product of a gene with sequence similarity to previously characterized bacterial porins.     

Common Duckweed (Lemna minor) Is a Versatile High-Throughput Infection Model For the Burkholderia cepacia Complex and Other Pathogenic Bacteria

Members of the Burkholderia cepacia complex (Bcc) have emerged in recent decades as problematic pulmonary pathogens of cystic fibrosis (CF) patients, with severe infections progressing to acute necrotizing pneumonia and sepsis. This study presents evidence that Lemna minor (Common duckweed) is useful as a plant model for the Bcc infectious process, and has potential as a model system for bacterial pathogenesis in general. To investigate the relationship between Bcc virulence in duckweed and Galleria mellonella (Greater wax moth) larvae, a previously established Bcc infection model, a duckweed survival assay was developed and used to determine LD₅₀ values. A strong correlation (R² = 0.81) was found between the strains’ virulence ranks in the two infection models, suggesting conserved pathways in these vastly different hosts. To broaden the application of the duckweed model, enteropathogenic Escherichia coli (EPEC) and five isogenic mutants with previously established LD₅₀ values in the larval model were tested against duckweed, and a strong correlation (R² = 0.93) was found between their raw LD₅₀ values. Potential virulence factors in B. cenocepacia K56-2 were identified using a high-throughput screen against single duckweed plants. In addition to the previously characterized antifungal compound (AFC) cluster genes, several uncharacterized genes were discovered including a novel lysR regulator, a histidine biosynthesis gene hisG, and a gene located near the gene encoding the recently characterized virulence factor SuhB_Bc. Finally, to demonstrate the utility of this model in therapeutic applications, duckweed was rescued from Bcc infection by treating with bacteriophage at 6-h intervals. It was observed that phage application became ineffective at a timepoint that coincided with a sharp increase in bacterial invasion of plant tissue. These results indicate that common duckweed can serve as an effective infection model for the investigation of bacterial virulence factors and therapeutic strategies to combat them.     

Antivirulence as a new antibacterial approach for chemotherapy

Bacterial resistance to antibiotics is an issue that has led to the search for new antibacterial approaches. Drugs targeting virulence is an alternative approach to treat infections due to resistant bacteria. There is extensive literature and knowledge in the field of bacterial pathogenesis and genomic determinant of virulence. As therapeutic targets, virulence factors have been primarily addressed in the vaccine field to prevent infection by specific pathogens. Recently novel strategies to identify virulence inhibitors have been numerous and several new compounds were recently reported. This review emphasizes the new virulence inhibitors that have shown a biological activity and have made a proof of concept that disarming bacteria lead to the inhibition of bacterial infection in experimental models in vivo. Moreover, some of these new antivirulence compounds are able to inhibit the virulence of different related pathogenic species, indicating that it is possible to target common virulence mechanisms. The progress reported recently with proof of concept for antivirulence molecules at the preclinical stages should allow the antivirulence concept to become a reality as a new antibacterial approach.     

Within-host competition in genetically diverse malaria infections: parasite virulence and competitive success

Humans and animals often become coinfected with pathogen strains that differ in virulence. The ensuing interaction between these strains can, in theory, be a major determinant of the direction of selection on virulence genes in pathogen populations. Many mathematical analyses of this assume that virulent pathogen lineages have a competitive advantage within coinfected hosts and thus predict that pathogens will evolve to become more virulent where genetically diverse infections are common. Although the implications of these studies are relevant to both fundamental biology and medical science, direct empirical tests for relationships between virulence and competitive ability are lacking. Here we use newly developed strain-specific real-time quantitative polymerase chain reaction protocols to determine the pairwise competitiveness of genetically divergent Plasmodium chabaudi clones that represent a wide range of innate virulences in their rodent host. We found that even against their background of widely varying genotypic and antigenic properties, virulent clones had a competitive advantage in the acute phase of mixed infections. The more virulent a clone was relative to its competitor, the less it suffered from competition. This result confirms our earlier work with parasite lines derived from a single clonal lineage by serial passage and supports the virulence-competitive ability assumption of many theoretical models. To the extent that our rodent model captures the essence of the natural history of malaria parasites, public health interventions which reduce the incidence of mixed malaria infections should have beneficial consequences by reducing the selection for high virulence.     

Sequence of Leptospira santarosai serovar Shermani genome and prediction of virulence-associated genes

Leptospirosis, a widespread zoonosis, is a re-emerging infectious disease caused by pathogenic Leptospira species. In Taiwan, Leptospira santarosai serovar Shermani is the most frequently isolated serovar, causing both renal and systemic infections. This study aimed to generate a L. santarosai serovar Shermani genome sequence and categorize its hypothetical genes, particularly those associated with virulence. The genome sequence consists of 3,936,333 nucleotides and 4033 predicted genes. Additionally, 2244 coding sequences could be placed into clusters of orthologous groups and the number of genes involving cell wall/membrane/envelope biogenesis and defense mechanisms was higher than that of other Leptospira spp. Comparative genetic analysis based on BLASTX data revealed that about 73% and 68.8% of all coding sequences have matches to pathogenic L. interrogans and L. borgpetersenii, respectively, and about 57.6% to saprophyte L. biflexa. Among the hypothetical proteins, 421 have a transmembrane region, 172 have a signal peptide and 17 possess a lipoprotein signature. According to PFAM prediction, 32 hypothetical proteins have properties of toxins and surface proteins mediated bacterial attachment, suggesting they may have roles associated with virulence. The availability of the genome sequence of L. santarosai serovar Shermani and the bioinformatics re-annotation of leptospiral hypothetical proteins will facilitate further functional genomic studies to elucidate the pathogenesis of leptospirosis and develop leptospiral vaccines.     

Genetic Adaptation of Achromobacter sp. during Persistence in the Lungs of Cystic Fibrosis Patients

Achromobacter species are increasingly isolated from the respiratory tract of cystic fibrosis patients and often a chronic infection is established. How Achromobacter sp. adapts to the human host remains uncharacterised. By comparing longitudinally collected isolates of Achromobacter sp. isolated from five CF patients, we have investigated the within-host evolution of clonal lineages. The majority of identified mutations were isolate-specific suggesting co-evolution of several subpopulations from the original infecting isolate. The largest proportion of mutated genes were involved in the general metabolism of the bacterium, but genes involved in virulence and antimicrobial resistance were also affected. A number of virulence genes required for initiation of acute infection were selected against, e.g. genes of the type I and type III secretion systems and genes related to pilus and flagellum formation or function. Six antimicrobial resistance genes or their regulatory genes were mutated, including large deletions affecting the repressor genes of an RND-family efflux pump and a beta-lactamase. Convergent evolution was observed for five genes that were all implicated in bacterial virulence. Characterisation of genes involved in adaptation of Achromobacter to the human host is required for understanding the pathogen-host interaction and facilitate design of future therapeutic interventions.