Genome evolution and the emergence of fruiting body development in Myxococcus xanthus

Background

Lateral gene transfer (LGT) is thought to promote speciation in bacteria, though well-defined examples have not been put forward.

Methodology/Principle Findings

We examined the evolutionary history of the genes essential for a trait that defines a phylogenetic order, namely fruiting body development of the Myxococcales. Seventy-eight genes that are essential for Myxococcus xanthus development were examined for LGT. About 73% of the genes exhibit a phylogeny similar to that of the 16S rDNA gene and a codon bias consistent with other M. xanthus genes suggesting vertical transmission. About 22% have an altered codon bias and/or phylogeny suggestive of LGT. The remaining 5% are unique. Genes encoding signal production and sensory transduction were more likely to be transmitted vertically with clear examples of duplication and divergence into multigene families. Genes encoding metabolic enzymes were frequently acquired by LGT. Myxobacteria exhibit aerobic respiration unlike most of the δ Proteobacteria. M. xanthus contains a unique electron transport pathway shaped by LGT of genes for succinate dehydrogenase and three cytochrome oxidase complexes.

Conclusions/Significance

Fruiting body development depends on genes acquired by LGT, particularly those involved in polysaccharide production. We suggest that aerobic growth fostered innovation necessary for development by allowing myxobacteria access to a different gene pool from anaerobic members of the δ Proteobacteria. Habitat destruction and loss of species diversity could restrict the evolution of new bacterial groups by limiting the size of the prospective gene pool.     

Identification of Anaplasma marginale type IV secretion system effector proteins

Background

Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.

Results

By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system.

Conclusions

The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.     

Transfer of energy pathway genes in microbial enhanced biological phosphorus removal communities

Background

Lateral gene transfer (LGT) is an important evolutionary process in microbial evolution. In sewage treatment plants, LGT of antibiotic resistance and xenobiotic degradation-related proteins has been suggested, but the role of LGT outside these processes is unknown. Microbial communities involved in Enhanced Biological Phosphorus Removal (EBPR) have been used to treat wastewater in the last 50 years and may provide insights into adaptation to an engineered environment. We introduce two different types of analysis to identify LGT in EBPR sewage communities, based on identifying assembled sequences with more than one strong taxonomic match, and on unusual phylogenetic patterns. We applied these methods to investigate the role of LGT in six energy-related metabolic pathways.

Results

The analyses identified overlapping but non-identical sets of transferred enzymes. All of these were homologous with sequences from known mobile genetic elements, and many were also in close proximity to transposases and integrases in the EBPR data set. The taxonomic method had higher sensitivity than the phylogenetic method, identifying more potential LGTs. Both analyses identified the putative transfer of five enzymes within an Australian community, two in a Danish community, and none in a US-derived culture.

Conclusions

Our methods were able to identify sequences with unusual phylogenetic or compositional properties as candidate LGT events. The association of these candidates with known mobile elements supports the hypothesis of transfer. The results of our analysis strongly suggest that LGT has influenced the development of functionally important energy-related pathways in EBPR systems, but transfers may be unique to each community due to different operating conditions or taxonomic composition.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1752-5) contains supplementary material, which is available to authorized users.     

Expression profiles of hydrophobic surfactant proteins in children with diffuse chronic lung disease

Background

Abnormalities of the intracellular metabolism of the hydrophobic surfactant proteins SP-B and SP-C and their precursors may be causally linked to chronic childhood diffuse lung diseases. The profile of these proteins in the alveolar space is unknown in such subjects.

Methods

We analyzed bronchoalveolar lavage fluid by Western blotting for SP-B, SP-C and their proforms in children with pulmonary alveolar proteinosis (PAP, n = 15), children with no SP-B (n = 6), children with chronic respiratory distress of unknown cause (cRD, n = 7), in comparison to children without lung disease (n = 15) or chronic obstructive bronchitis (n = 19).

Results

Pro-SP-B of 25–26 kD was commonly abundant in all groups of subjects, suggesting that their presence is not of diagnostic value for processing defects. In contrast, pro-SP-B peptides cleaved off during intracellular processing of SP-B and smaller than 19–21 kD, were exclusively found in PAP and cRD. In 4 of 6 children with no SP-B, mutations of SFTPB or SPTPC genes were found. Pro-SP-C forms were identified at very low frequency. Their presence was clearly, but not exclusively associated with mutations of the SFTPB and SPTPC genes, impeding their usage as candidates for diagnostic screening.

Conclusion

Immuno-analysis of the hydrophobic surfactant proteins and their precursor forms in bronchoalveolar lavage is minimally invasive and can give valuable clues for the involvement of processing abnormalities in pediatric pulmonary disorders.     

Induction of Interferon-Stimulated Genes by Chlamydia pneumoniae in Fibroblasts Is Mediated by Intracellular Nucleotide-Sensing Receptors

Background

Recognition of microorganisms by the innate immune system is mediated by pattern recognition receptors, including Toll-like receptors and cytoplasmic RIG-I-like receptors. Chlamydia, which include several human pathogenic species, are obligate intracellular gram-negative bacteria that replicate in cytoplasmic vacuoles. The infection triggers a host response contributing to both bacterial clearance and tissue damage. For instance, type I interferons (IFN)s have been demonstrated to exacerbate the course of Chlamydial lung infections in mice.

Methods/Principal Findings

Here we show that Chlamydia pneumoniae induces expression of IFN-stimulated genes (ISG)s dependent on recognition by nucleotide-sensing Toll-like receptors and RIG-I-like receptors, localized in endosomes and the cytoplasm, respectively. The ISG response was induced with a delayed kinetics, compared to virus infections, and was dependent on bacterial replication and the bacterial type III secretion system (T3SS).

Conclusions/Significance

Activation of the IFN response during C. pneumoniae infection is mediated by intracellular nucleotide-sensing PRRs, which operate through a mechanism dependent on the bacterial T3SS. Strategies to inhibit the chlamydial T3SS may be used to limit the detrimental effects of the type I IFN system in the host response to Chlamydia infection.     

Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis

Background

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is an important gene regulation process that potentially regulates many physiological processes in plants, including the response to abiotic stresses such as salt stress.

Results

To analyze global changes in AS under salt stress, we obtained high-coverage (~200 times) RNA sequencing data from Arabidopsis thaliana seedlings that were treated with different concentrations of NaCl. We detected that ~49% of all intron-containing genes were alternatively spliced under salt stress, 10% of which experienced significant differential alternative splicing (DAS). Furthermore, AS increased significantly under salt stress compared with under unstressed conditions. We demonstrated that most DAS genes were not differentially regulated by salt stress, suggesting that AS may represent an independent layer of gene regulation in response to stress. Our analysis of functional categories suggested that DAS genes were associated with specific functional pathways, such as the pathways for the responses to stresses and RNA splicing. We revealed that serine/arginine-rich (SR) splicing factors were frequently and specifically regulated in AS under salt stresses, suggesting a complex loop in AS regulation for stress adaptation. We also showed that alternative splicing site selection (SS) occurred most frequently at 4 nucleotides upstream or downstream of the dominant sites and that exon skipping tended to link with alternative SS.

Conclusions

Our study provided a comprehensive view of AS under salt stress and revealed novel insights into the potential roles of AS in plant response to salt stress.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-431) contains supplementary material, which is available to authorized users.     

ACE as a mechanosensor to shear stress influences the control of its own regulation via phosphorylation of cytoplasmic Ser(1270)

Objectives

We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser¹²⁷⁰ are involved in shear-stress (SS)-induced downregulation of the enzyme.

Methods and Results

Western blotting analysis showed that SS (18 h, 15 dyn/cm²) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra- or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser¹²⁷⁰ compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor.

Conclusions

ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser¹²⁷⁰, consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser¹²⁷⁰.     

Amoebae as Potential Environmental Hosts for Mycobacterium ulcerans and Other Mycobacteria, but Doubtful Actors in Buruli Ulcer Epidemiology

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, remain unknown. Ecological, genetic and epidemiological information nonetheless suggests that M. ulcerans may reside in aquatic protozoa.

Methodology/Principal Findings

We experimentally infected Acanthamoeba polyphaga with M. ulcerans and found that the bacilli were phagocytised, not digested and remained viable for the duration of the experiment. Furthermore, we collected 13 water, 90 biofilm and 45 detritus samples in both Buruli ulcer endemic and non-endemic communities in Ghana, from which we cultivated amoeboid protozoa and mycobacteria. M. ulcerans was not isolated, but other mycobacteria were as frequently isolated from intracellular as from extracellular sources, suggesting that they commonly infect amoebae in nature. We screened the samples as well as the amoeba cultures for the M. ulcerans markers IS2404, IS2606 and KR-B. IS2404 was detected in 2% of the environmental samples and in 4% of the amoeba cultures. The IS2404 positive amoeba cultures included up to 5 different protozoan species, and originated both from Buruli ulcer endemic and non-endemic communities.

Conclusions/Significance

This is the first report of experimental infection of amoebae with M. ulcerans and of the detection of the marker IS2404 in amoeba cultures isolated from the environment. We conclude that amoeba are potential natural hosts for M. ulcerans, yet remain sceptical about their implication in the transmission of M. ulcerans to humans and their importance in the epidemiology of Buruli ulcer.     

Acanthamoeba polyphaga-enhanced growth of Mycobacterium smegmatis

Background

Mycobacterium smegmatis is a rapidly-growing mycobacterium causing rare opportunistic infections in human patients. It is present in soil and water environments where free-living amoeba also reside, but data regarding M. smegmatis-amoeba relationships have been contradictory from mycobacteria destruction to mycobacteria survival.

Methodology/Principal Findings

Using optic and electron microscopy and culture-based microbial enumeration we investigated the ability of M. smegmatis mc² 155, M. smegmatis ATCC 19420^T and M. smegmatis ATCC 27204 organisms to survive into Acanthamoeba polyphaga trophozoites and cysts. We observed that M. smegmatis mycobacteria penetrated and survived in A. polyphaga trophozoites over five-day co-culture resulting in amoeba lysis and the release of viable M. smegmatis mycobacteria without amoebal cyst formation. We further observed that amoeba-co-culture, and lysed amoeba and supernatant and pellet, significantly increased five-day growth of the three tested M. smegmatis strains, including a four-fold increase in intra-amoebal growth.

Conclusions/Significance

Amoebal co-culture increases the growth of M. smegmatis resulting in amoeba killing by replicating M. smegmatis mycobacteria. This amoeba-M. smegmatis co-culture system illustrates an unusual paradigm in the mycobacteria-amoeba interactions as mycobacteria have been mainly regarded as amoeba-resistant organisms. Using these model organisms, this co-culture system could be used as a simple and rapid model to probe mycobacterial factors implicated in the intracellular growth of mycobacteria.     

Lipid metabolism and Type VII secretion systems dominate the genome scale virulence profile of Mycobacterium tuberculosis in human dendritic cells

Background

Mycobacterium tuberculosis continues to kill more people than any other bacterium. Although its archetypal host cell is the macrophage, it also enters, and survives within, dendritic cells (DCs). By modulating the behaviour of the DC, M. tuberculosis is able to manipulate the host’s immune response and establish an infection. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive.

Results

Parallel sequencing of the transposon inserts of the surviving mutants identified a large number of genes as being required for optimal intracellular fitness in DCs. Loci whose mutation attenuated intracellular survival included those involved in synthesising cell wall lipids, not only the well-established virulence factors, pDIM and cord factor, but also sulfolipids and PGL, which have not previously been identified as having a direct virulence role in cells. Other attenuated loci included the secretion systems ESX-1, ESX-2 and ESX-4, alongside many PPE genes, implicating a role for ESX-5. In contrast the canonical ESAT-6 family of ESX substrates did not have intra-DC fitness costs suggesting an alternative ESX-1 associated virulence mechanism. With the aid of a gene-nutrient interaction model, metabolic processes such as cholesterol side chain catabolism, nitrate reductase and cysteine-methionine metabolism were also identified as important for survival in DCs.

Conclusion

We conclude that many of the virulence factors required for survival in DC are shared with macrophages, but that survival in DCs also requires several additional functions, such as cysteine-methionine metabolism, PGLs, sulfolipids, ESX systems and PPE genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1569-2) contains supplementary material, which is available to authorized users.     

Characterization of clinically-attenuated Burkholderia mallei by whole genome sequencing: candidate strain for exclusion from Select Agent lists

Background

Burkholderia mallei is an understudied biothreat agent responsible for glanders which can be lethal in humans and animals. Research with this pathogen has been hampered in part by constraints of Select Agent regulations for safety reasons. Whole genomic sequencing (WGS) is an apt approach to characterize newly discovered or poorly understood microbial pathogens.

Methodology/Principal Findings

We performed WGS on a strain of B. mallei, SAVP1, previously pathogenic, that was experimentally infected in 6 equids (4 ponies, 1 mule, 1 donkey), natural hosts, for purposes of producing antibodies. Multiple high inocula were used in some cases. Unexpectedly SAVP1 appeared to be avirulent in the ponies and mule, and attenuated in the donkey, but induced antibodies. We determined the genome sequence of SAVP1 and compared it to a strain that was virulent in horses and a human. In comparison, this phenotypic avirulent SAVP1 strain was missing multiple genes including all the animal type III secretory system (T3SS) complex of genes demonstrated to be essential for virulence in mice and hamster models. The loss of these genes in the SAVP1 strain appears to be the consequence of a multiple gene deletion across insertion sequence (IS) elements in the B. mallei genome. Therefore, the strain by itself is unlikely to revert naturally to its virulent phenotype. There were other genes present in one strain and not the other and vice-versa.

Conclusion/Significance

The discovery that this strain of B. mallei was both avirulent in the natural host ponies, and did not possess T3SS associated genes may be fortuitous to advance biodefense research. The deleted virulence-essential T3SS is not likely to be re-acquired naturally. These findings may provide a basis for exclusion of SAVP1 from the Select Agent regulation or at least discussion of what else would be required for exclusion. This exclusion could accelerate research by investigators not possessing BSL-3 facilities and facilitate the production of reagents such as antibodies without the restraints of Select Agent regulation.     

Extensive duplication of the Wolbachia DNA in chromosome four of Drosophila ananassae

Background

Lateral gene transfer (LGT) from bacterial Wolbachia endosymbionts has been detected in ~20% of arthropod and nematode genome sequencing projects. Many of these transfers are large and contain a substantial part of the Wolbachia genome.

Results

Here, we re-sequenced three D. ananassae genomes from Asia and the Pacific that contain large LGTs from Wolbachia. We find that multiple copies of the Wolbachia genome are transferred to the Drosophila nuclear genome in all three lines. In the D. ananassae line from Indonesia, the copies of Wolbachia DNA in the nuclear genome are nearly identical in size and sequence yielding an even coverage of mapped reads over the Wolbachia genome. In contrast, the D. ananassae lines from Hawaii and India show an uneven coverage of mapped reads over the Wolbachia genome suggesting that different parts of these LGTs are present in different copy numbers. In the Hawaii line, we find that this LGT is underrepresented in third instar larvae indicative of being heterochromatic. Fluorescence in situ hybridization of mitotic chromosomes confirms that the LGT in the Hawaii line is heterochromatic and represents ~20% of the sequence on chromosome 4 (dot chromosome, Muller element F).

Conclusions

This collection of related lines contain large lateral gene transfers composed of multiple Wolbachia genomes that constitute >2% of the D. ananassae genome (~5 Mbp) and partially explain the abnormally large size of chromosome 4 in D. ananassae.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1097) contains supplementary material, which is available to authorized users.     

Eradication of intracellular Francisella tularensis in THP-1 human macrophages with a novel autophagy inducing agent

Background

Autophagy has been shown recently to play an important role in the intracellular survival of several pathogenic bacteria. In this study, we investigated the effect of a novel small-molecule autophagy-inducing agent, AR-12, on the survival of Francisella tularensis, the causative bacterium of tularemia in humans and a potential bioterrorism agent, in macrophages.

Methods and results

Our results show that AR-12 induces autophagy in THP-1 macrophages, as indicated by increased autophagosome formation, and potently inhibits the intracellular survival of F. tularensis (type A strain, Schu S4) and F. novicida in macrophages in association with increased bacterial co-localization with autophagosomes. The effect of AR-12 on intracellular F. novicida was fully reversed in the presence of the autophagy inhibitor, 3-methyl adenine or the lysosome inhibitor, chloroquine. Intracellular F. novicida were not susceptible to the inhibitory activity of AR-12 added at 12 h post-infection in THP-1 macrophages, and this lack of susceptibility was independent of the intracellular location of bacteria.

Conclusion

Together, AR-12 represents a proof-of-principle that intracellular F. tularensis can be eradicated by small-molecule agents that target innate immunity.     

Impaired Clearance of Early Apoptotic Cells Mediated by Inhibitory IgG Antibodies in Patients with Primary Sj?gren's Syndrome

Objectives

Deficient efferocytosis (i.e. phagocytic clearance of apoptotic cells) has been frequently reported in systemic lupus erythematosus (SLE). Todate, patients with primary Sjögren''s syndrome (SS) have not been assessed for phagocytosis of apoptotic cells (ApoCell-phagocytosis) and of particulate targets (microbeads, MB-phagocytosis).

Design

ApoCell-phagocytosis and MB-phagocytosis were comparatively assessed by flow cytometry in peripheral blood specimens and monocyte-derived macrophage (MDM) preparations from healthy blood donors (HBD) and consecutive SS, SLE and rheumatoid arthritis (RA) patients. Cross-admixture ApoCell-phagocytosis experiments were also performed using phagocytes from HBD or patients, and apoptotic cells pretreated with whole sera or purified serum IgG derived from patients or HBD.

Results

Compared to HBD, approximately half of SS and SLE patients studied (but not RA) manifested significantly reduced ApoCell-phagocytosis (p<0.001) and MB-phagocytosis (p<0.003) by blood-borne phagocytes that correlated inversely with disease activity (p≤0.004). In cross-admixture assays, healthy monocytes showed significantly reduced ApoCell-phagocytosis when fed with apoptotic cells that were pretreated with sera or purified serum IgG preparations from SS and SLE patients (p<0.0001, compared to those from HBD or RA). Such aberrant effect of the SS and SLE sera and IgG preparations correlated linearly with their content of IgG antibodies against apoptotic cells (p≤0.0001). Phagocytic dysfunction maybe also present in certain SS and SLE patients, as supported by deficient capacity of MDM for ApoCell-phagocytosis and MB-phagocytosis under patients'' serum-free conditions.

Conclusion

Similarly to SLE, efferocytosis is frequently impaired in SS and is primarily due to the presence of inhibitory IgG anti-ApoCell antibodies and secondarily to phagocytes'' dysfunction.