Iron-regulated small RNA expression as Neisseria gonorrhoeae FA 1090 transitions into stationary phase growth.

BACKGROUND: For most pathogens, iron (Fe) homeostasis is crucial for maintenance within the host and the ability to cause disease. The primary transcriptional regulator that controls intracellular Fe levels is the Fur (ferric uptake regulator) protein, which exerts its action on transcription by binding to a promoter-proximal sequence termed the Fur box. Fur-regulated transcriptional responses are often fine-tuned at the post-transcriptional level through the action of small regulatory RNAs (sRNAs). Consequently, identifying sRNAs contributing to the control of Fe homeostasis is important for understanding the Fur-controlled bacterial Fe-response network. RESULTS: In this study, we sequenced size-selected directional libraries representing sRNA samples from Neisseria gonorrhoeae strain FA 1090, and examined the Fe- and temporal regulation of these sRNAs. RNA-seq data for all time points identified a pool of at least 340 potential sRNAs. Differential analysis demonstrated that expression appeared to be regulated by Fe availability for at least fifteen of these sRNAs. Fourteen sRNAs were induced in high Fe conditions, consisting of both cis and trans sRNAs, some of which are predicted to control expression of a known virulence factor, and one SAM riboswitch. An additional putative cis-acting sRNA was repressed by Fe availability. In the pathogenic Neisseria species, one sRNA that contributes to Fe-regulated post-transcriptional control is the Fur-repressible sRNA NrrF. The expression of five Fe-induced sRNAs appeared to be at least partially controlled by NrrF, while the remainder was expressed independently of NrrF. The expression of the 14 Fe-induced sRNAs also exhibited temporal control, as their expression levels increased dramatically as the bacteria entered stationary phase. CONCLUSIONS: Here we report the temporal expression of Fe-regulated sRNAs in N. gonorrhoeae FA 1090 with several appearing to be controlled by the Fe-repressible sRNA NrrF. Temporal regulation of these sRNAs suggests a regulatory role in controlling functions necessary for survival, and may be important for phenotypes often associated with altered growth rates, such as biofilm formation or intracellular survival. Future functional studies will be needed to understand how these regulatory sRNAs contribute to gonococcal biology and pathogenesis.

Phosphorylation within the cysteine-rich region of dystrophin enhances its association with ß-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting.

Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin-glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin-dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin.

Consensus computational network analysis for identifying candidate outer membrane proteins from Borrelia spirochetes.

BACKGROUND: Similar to Gram-negative organisms, Borrelia spirochetes are dual-membrane organisms with both an inner and outer membrane. Although the outer membrane contains integral membrane proteins, few of the borrelial outer membrane proteins (OMPs) have been identified and characterized to date. Therefore, we utilized a consensus computational network analysis to identify novel borrelial OMPs. RESULTS: Using a series of computer-based algorithms, we selected all protein-encoding sequences predicted to be OM-localized and/or to form ß-barrels in the borrelial OM. Using this system, we identified 41 potential OMPs from B. burgdorferi and characterized three (BB0838, BB0405, and BB0406) to confirm that our computer-based methodology did, in fact, identify borrelial OMPs. Triton X-114 phase partitioning revealed that BB0838 is found in the detergent phase, which would be expected of a membrane protein. Proteolysis assays indicate that BB0838 is partially sensitive to both proteinase K and trypsin, further indicating that BB0838 is surface-exposed. Consistent with a prior study, we also confirmed that BB0405 is surface-exposed and associates with the borrelial OM. Furthermore, we have shown that BB0406, the product of a co-transcribed downstream gene, also encodes a novel, previously uncharacterized borrelial OMP. Interestingly, while BB0406 has several physicochemical properties consistent with it being an OMP, it was found to be resistant to surface proteolysis. Consistent with BB0405 and BB0406 being OMPs, both were found to be capable of incorporating into liposomes and exhibit pore-forming activity, suggesting that both proteins are porins. Lastly, we expanded our computational analysis to identify OMPs from other borrelial organisms, including both Lyme disease and relapsing fever spirochetes. CONCLUSIONS: Using a consensus computer algorithm, we generated a list of candidate OMPs for both Lyme disease and relapsing fever spirochetes and determined that three of the predicted B. burgdorferi proteins identified were indeed novel borrelial OMPs. The combined studies have identified putative spirochetal OMPs that can now be examined for their roles in virulence, physiology, and disease pathogenesis. Importantly, the studies described in this report provide a framework by which OMPs from any human pathogen with a diderm ultrastructure could be cataloged to identify novel virulence factors and vaccine candidates.

Divergence of the SigB regulon and pathogenesis of the Bacillus cereus sensu lato group.

BACKGROUND: The Bacillus cereus sensu lato group currently includes seven species (B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus) that recent phylogenetic and phylogenomic analyses suggest are likely a single species, despite their varied phenotypes. Although horizontal gene transfer and insertion-deletion events are clearly important for promoting divergence among these genomes, recent studies have demonstrated that a major basis for phenotypic diversity in these organisms may be differential regulation of the highly similar gene content shared by these organisms. To explore this hypothesis, we used an in silico approach to evaluate the relationship of pathogenic potential and the divergence of the SigB-dependent general stress response within the B. cereus sensu lato group, since SigB has been demonstrated to support pathogenesis in Bacillus, Listeria and Staphylococcus species. RESULTS: During the divergence of these organisms from a common "SigB-less" ancestor, the placement of SigB promoters at varied locations in the B. cereus sensu lato genomes predict alternative structures for the SigB regulon in different organisms. Predicted promoter changes suggesting differential transcriptional control of a common gene pool predominate over evidence of indels or horizontal gene transfer for explaining SigB regulon divergence. CONCLUSIONS: Four lineages of the SigB regulon have arisen that encompass different gene contents and suggest different strategies for supporting pathogenesis. This is consistent with the hypothesis that divergence within the B. cereus sensu lato group rests in part on alternative strategies for regulation of a common gene pool.

Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group.

BACKGROUND: The Bacillus cereus sensu lato group consists of six species (B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. weihenstephanensis). While classical microbial taxonomy proposed these organisms as distinct species, newer molecular phylogenies and comparative genome sequencing suggests that these organisms should be classified as a single species (thus, we will refer to these organisms collectively as the Bc species-group). How do we account for the underlying similarity of these phenotypically diverse microbes? It has been established for some time that the most rapidly evolving and evolutionarily flexible portions of the bacterial genome are regulatory sequences and transcriptional networks. Other studies have suggested that the sigma factor gene family of these organisms has diverged and expanded significantly relative to their ancestors; sigma factors are those portions of the bacterial transcriptional apparatus that control RNA polymerase recognition for promoter selection. Thus, examining sigma factor divergence in these organisms would concurrently examine both regulatory sequences and transcriptional networks important for divergence. We began this examination by comparison to the sigma factor gene set of B. subtilis. RESULTS: Phylogenetic analysis of the Bc species-group utilizing 157 single-copy genes of the family Bacillaceae suggests that several taxonomic revisions of the genus Bacillus should be considered. Within the Bc species-group there is little indication that the currently recognized species form related sub-groupings, suggesting that they are members of the same species. The sigma factor gene family encoded by the Bc species-group appears to be the result of a dynamic gene-duplication and gene-loss process that in previous analyses underestimated the true heterogeneity of the sigma factor content in the Bc species-group. CONCLUSIONS: Expansion of the sigma factor gene family appears to have preferentially occurred within the extracytoplasmic function (ECF) sigma factor genes, while the primary alternative (PA) sigma factor genes are, in general, highly conserved with those found in B. subtilis. Divergence of the sigma-controlled transcriptional regulons among various members of the Bc species-group likely has a major role in explaining the diversity of phenotypic characteristics seen in members of the Bc species-group.

Human C. difficile toxin-specific memory B cell repertoires encode poorly neutralizing antibodies.

Clostridioides difficile is a leading cause of nosocomial infection responsible for significant morbidity and mortality with limited options for therapy. Secreted C. difficile toxin B (TcdB) is a major contributor to disease pathology, and select TcdB-specific Abs may protect against disease recurrence. However, the high frequency of recurrence suggests that the memory B cell response, essential for new Ab production following C. difficile reexposure, is insufficient. We therefore isolated TcdB-specific memory B cells from individuals with a history of C. difficile infection and performed single-cell deep sequencing of their Ab genes. Herein, we report that TcdB-specific memory B cell-encoded antibodies showed somatic hypermutation but displayed limited isotype class switch. Memory B cell-encoded mAb generated from the gene sequences revealed low to moderate affinity for TcdB and a limited ability to neutralize TcdB. These findings indicate that memory B cells are an important factor in C. difficile disease recurrence.

Draft Genome Sequence of Pediatric Otitis Media Isolate Streptococcus pneumoniae Strain EF3030, Which Forms In Vitro Biofilms That Closely Mimic In Vivo Biofilms.

Here, we report the draft genome sequence of Streptococcus pneumoniae EF3030, a pediatric otitis media isolate active in biofilm assays of epithelial colonization. The final draft assembly included 2,209,198 bp; the annotation predicted 2,120 coding DNA sequences (CDSs), 4 complete rRNA operons, 58 tRNAs, 3 noncoding RNAs (ncRNAs), and 199 pseudogenes.

Single-synapse ablation and long-term imaging in live C. elegans.

Synapses are individually operated, computational units for neural communication. To manipulate physically individual synapses in a living organism, we have developed a laser ablation technique for removing single synapses in live neurons in C. elegans that operates without apparent damage to the axon. As a complementary technique, we applied microfluidic immobilization of C. elegans to facilitate long-term fluorescence imaging and observation of neuronal development. With this technique, we directly demonstrated the existence of competition between developing synapses in the HSNL motor neuron.

Route of infection alters virulence of neonatal septicemia Escherichia coli clinical isolates.

Escherichia coli is the leading cause of Gram-negative neonatal septicemia in the United States. Invasion and passage across the neonatal gut after ingestion of maternal E. coli strains produce bacteremia. In this study, we compared the virulence properties of the neonatal E. coli bacteremia clinical isolate SCB34 with the archetypal neonatal E. coli meningitis strain RS218. Whole-genome sequencing data was used to compare the protein coding sequences among these clinical isolates and 33 other representative E. coli strains. Oral inoculation of newborn animals with either strain produced septicemia, whereas intraperitoneal injection caused septicemia only in pups infected with RS218 but not in those injected with SCB34. In addition to being virulent only through the oral route, SCB34 demonstrated significantly greater invasion and transcytosis of polarized intestinal epithelial cells in vitro as compared to RS218. Protein coding sequences comparisons highlighted the presence of known virulence factors that are shared among several of these isolates, and revealed the existence of proteins exclusively encoded in SCB34, many of which remain uncharacterized. Our study demonstrates that oral acquisition is crucial for the virulence properties of the neonatal bacteremia clinical isolate SCB34. This characteristic, along with its enhanced ability to invade and transcytose intestinal epithelium are likely determined by the specific virulence factors that predominate in this strain.

Small-Molecule Inhibition of GCNT3 Disrupts Mucin Biosynthesis and Malignant Cellular Behaviors in Pancreatic Cancer.

Pancreatic cancer is an aggressive neoplasm with almost uniform lethality and a 5-year survival rate of 7%. Several overexpressed mucins that impede drug delivery to pancreatic tumors have been therapeutically targeted, but enzymes involved in mucin biosynthesis have yet to be preclinically evaluated as potential targets. We used survival data from human patients with pancreatic cancer, next-generation sequencing of genetically engineered Kras-driven mouse pancreatic tumors and human pancreatic cancer cells to identify the novel core mucin-synthesizing enzyme GCNT3 (core 2 ß-1,6 N-acetylglucosaminyltransferase). In mouse pancreatic cancer tumors, GCNT3 upregulation (103-fold; P < 0.0001) was correlated with increased expression of mucins (5 to 87-fold; P < 0.04-0.0003). Aberrant GCNT3 expression was also associated with increased mucin production, aggressive tumorigenesis, and reduced patient survival, and CRISPR-mediated knockout of GCNT3 in pancreatic cancer cells reduced proliferation and spheroid formation. Using in silico small molecular docking simulation approaches, we identified talniflumate as a novel inhibitor that selectively binds to GCNT3. In particular, docking predictions suggested that three notable hydrogen bonds between talniflumate and GCNT3 contribute to a docking affinity of -8.3 kcal/mol. Furthermore, talniflumate alone and in combination with low-dose gefitinib reduced GCNT3 expression, leading to the disrupted production of mucins in vivo and in vitro Collectively, our findings suggest that targeting mucin biosynthesis through GCNT3 may improve drug responsiveness, warranting further development and investigation in preclinical models of pancreatic tumorigenesis. Cancer Res; 76(7); 1965-74. ©2016 AACR.