Francisella tularensis is a gram-negative pathogen that causes life-threatening infections in humans and has potential for use as a biological weapon. The genetic basis of the F. tularensis virulence is poorly understood. This study screened a total of 3,936 transposon mutants of the live vaccine strain for infection in a mouse model of respiratory tularemia by signature-tagged mutagenesis. We identified 341 mutants attenuated for infection in the lungs. The transposon disruptions were mapped to 95 different genes, virtually all of which are also present in the genomes of other F. tularensis strains, including human pathogenic F. tularensis strain Schu S4. A small subset of these attenuated mutants carried insertions in the genes encoding previously known virulence factors, but the majority of the identified genes have not been previously linked to F. tularensis virulence. Among these are genes encoding putative membrane proteins, proteins associated with stress responses, metabolic proteins, transporter proteins, and proteins with unknown functions. Several attenuated mutants contained disruptions in a putative capsule locus which partially resembles the poly-␥-glutamate capsule biosynthesis locus of Bacillus anthracis, the anthrax agent. Deletional mutation analysis confirmed that this locus is essential for F. tularensis virulence.

c Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology and pathogenesis. However, these nucleotides have not been explored in Streptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes of S. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth, resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is essential for pneumococcal biology and disease.

Summary Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. The pathogenesis by the causative agent, Mycobacterium tuberculosis, is still not fully understood. We have previously reported that M. tuberculosis Rv3586 (disA) encodes a diadenylate cyclase, which converts ATP to cyclic di-AMP (c-di-AMP). In this study, we demonstrated that a protein encoded by Rv2837c (cnpB) possesses c-di-AMP phosphodiesterase activity and cleaves c-di-AMP exclusively to AMP. Our results showed that in M. tuberculosis, deletion of disA abolished bacterial c-di-AMP production, whereas deletion of cnpB significantly enhanced the bacterial c-di-AMP accumulation and secretion. The c-di-AMP levels in both mutants could be corrected by expressing the respective gene. We also found that macrophages infected with ΔcnpB secreted much higher levels of IFN-β than those infected with the wildtype (WT) or the complemented mutant. Interestingly, mice infected with M. tuberculosis ΔcnpB displayed significantly reduced inflammation, less bacterial burden in the lungs and spleens, and extended survival compared to those infected with the WT or the complemented mutant. These results indicate that deletion of cnpB results in attenuated virulence, which is correlated with elevated c-di-AMP levels.

Cyclic di-AMP (c-di-AMP) has been shown to play important roles as a second messenger in bacterial physiology and infections. However, understanding of how the signal is transduced is still limited. Previously, we have characterized a diadenylate cyclase and two c-di-AMP phosphodiesterases in Streptococcus pneumoniae, a Gram-positive pathogen. In this study, we identified a c-di-AMP binding protein (CabP) in S. pneumoniae using c-di-AMP affinity chromatography. We demonstrated that CabP specifically bound c-di-AMP and that this interaction could not be interrupted by competition with other nucleotides, including ATP, cAMP, AMP, phosphoadenylyl adenosine (pApA), and cyclic di-GMP (c-di-GMP). By using a bacterial two-hybrid system and genetic mutagenesis, we showed that CabP directly interacted with a potassium transporter (SPD_0076) and that both proteins were required for pneumococcal growth in media with low concentrations of potassium. Interestingly, the interaction between CabP and SPD_0076 and the efficiency of potassium uptake were impaired by elevated c-di-AMP in pneumococci. These results establish a direct c-di-AMP-mediated signaling pathway that regulates pneumococcal potassium uptake.

Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg2+, Mn2+ or Co2+. DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis.

Variation of maternal gut microbiota may increase the risk of autism spectrum disorders (ASDs) in offspring. Animal studies have indicated that maternal gut microbiota is related to neurodevelopmental abnormalities in mouse offspring, while it is unclear whether there is a correlation between gut microbiota of ASD children and their mothers. We examined the relationships between gut microbiome profiles of ASD children and those of their mothers, and evaluated the clinical discriminatory power of discovered bacterial biomarkers . Gut microbiome was profiled and evaluated by 16S ribosomal RNA gene sequencing in stool samples of 59 mother–child pairs of ASD children and 30 matched mother–child pairs of healthy children. Significant differences were observed in the gut microbiome composition between ASD and healthy children in our Chinese cohort. Several unique bacterial biomarkers, such as Alcaligenaceae and Acinetobacter , were identified. Mothers of ASD children had more Proteobacteria, Alphaproteobacteria, Moraxellaceae, and Acinetobacter than mothers of healthy children. There was a clear correlation between gut microbiome profiles of children and their mothers; however, children with ASD still had unique bacterial biomarkers, such as Alcaligenaceae, Enterobacteriaceae, and Clostridium . Candidate biomarkers discovered in this study had remarkable discriminatory power. The identified patterns of mother–child gut microbiome profiles may be important for assessing risks during the early stage and planning of personalized treatment and prevention of ASD via microbiota modulation.

Vitamin B 6 is an essential cofactor for a large number of enzymes in both prokaryotes and eukaryotes. In this study, we characterized the pyridoxal 5=-phosphate (PLP) biosynthesis pathway in Streptococcus pneumoniae. Our results revealed that S. pneumoniae possesses a de novo vitamin B 6 biosynthesis pathway encoded by the pdxST genes. Purified PdxS functionally displayed as PLP synthase, whereas PdxT exhibited glutaminase activity in vitro. Deletion of pdxS, but not pdxT, resulted in a vitamin B 6 auxotrophic mutant. The defective growth of the ⌬pdxS mutant in a vitamin B 6 -depleted medium could be chemically restored in the presence of the B 6 vitamers at optimal concentrations. By analyzing PdxS expression levels, we demonstrated that the expression of pdxS was repressed by PLP and activated by a transcription factor, PdxR. A pneumococcal ⌬pdxR mutant also exhibited as a vitamin B 6 auxotroph. In addition, we found that disruption of the vitamin B 6 biosynthesis pathway in S. pneumoniae caused a significant attenuation in a chinchilla middle ear infection model and a minor attenuation in a mouse pneumonia model, indicating that the impact of vitamin B 6 synthesis on virulence depends upon the bacterial infection niche.

BackgroundEar infection or otitis media (OM) accounts for most bacterial respiratory infections in children in both developed and developing nations. Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis are the major OM pathogens. However, little is known about the genetic basis of bacterial OM largely due to practical difficulties in conducting research in ear infection models and genetically manipulating clinical isolates. Here, we report the first genome-scale in vivo screen for bacterial genes required for ear infection in a chinchilla model by signature tagged mutagenesis (STM), a high throughput mutant screen technique.Methodology/Principal FindingsSTM strains were constructed with a multi-drug resistant OM isolate ST556 (serotype 19F) and screened in a chinchilla OM model. Out of 5,280 mutants tested, 248 mutants were substantially underrepresented in the mutant pools recovered from the middle ear fluids of the infected chinchillas, indicating the impaired ability to survive and replicate in the middle ears due to genetic disruptions in the chromosome of strain ST556. Further DNA sequencing analysis mapped the mutations to 169 pneumococcal genes. Surprisingly, only 52 of these genes were required for pneumococcal nasopharyngeal colonization in a murine model. This infection site-specific gene requirement was verified by targeted mutagenesis in the selected genes.Conclusions/SignificanceThese findings suggest that there are a subset of pneumococcal genes required for ear infection and that these may be distinct from those required for nasal colonization. Our data thus provide comprehensive gene targets for mechanistic understanding of pneumococcal ear infection. Finally, this study has also developed a model for future genome-scale search for virulence determinants in other pathogens associated with ear infections.