Effect of 3-Amino-1,2,4-Triazole on Histidine Metabolism in Algae

Growth of Chlorella vulgaris and Prototheca zopfii is inhibited by treatment with 3-amino-1,2,4-triazole. Growth of these 2 algae in the presence of amino triazole is accompanied by the accumulation in the culture media of the dephosphorylated histidine precursor, imidazoleglycerol. The addition of histidine to the culture media in the presence of amino triazole restored the normal growth rates of these organisms and reduced the accumulation of imidazoleglycerol. These data suggest that amino triazole inhibits the synthesis of histidine by interfering with the activity of the enzyme, imidazoleglycerol phosphate dehydrase.     

Dual signaling functions of the hybrid sensor kinase RpfC of Xanthomonas campestris involve either phosphorelay or receiver domain-protein interaction

The hybrid sensor kinase RpfC positively regulates the expression of a range of virulent genes and negatively modulates the synthesis of the quorum sensing signal diffusible signal factor (DSF) in Xanthomonas campestris. Three conserved amino acid residues of RpfC implicated in phosphorelay (His(198) in the histidine kinase domain, Asp(512) in the receiver domain, and His(657) in the histidine phosphotransfer domain) were essential for activation of the production of extracellular enzymes and extracellular polysaccharide (EPS) virulence factors but were not essential for repression of DSF biosynthesis. Domain deletion and subsequent in trans expression analysis revealed that the receiver domain of RpfC alone was sufficient to repress DSF overproduction in an rpfC deletion mutant. Further deletion and alanine scanning mutagenesis analyses identified a peptide of 107 amino acids and three amino acid residues (Gln(496), Glu(504), and Ile(552)) involved in modulating DSF production. Co-immunoprecipitation and far Western blot analyses suggested an interaction between the receiver domain and RpfF, the enzyme involved in DSF biosynthesis. These data support a model in which RpfC modulates two different functions (virulence factor synthesis and DSF synthesis) by utilization of a conserved phosphorelay system and a novel domain-specific protein-protein interaction mechanism, respectively. This latter mechanism represents an added dimension to conventional two-component signaling paradigms.     

Crystallization and initial X-ray diffraction analysis of the multi-domain Brucella blue light-activated histidine kinase LOV-HK in its illuminated state

The pathogenic bacterium Brucella abortus codes for a multi-domain dimeric cytoplasmic histidine kinase called LOV-HK, which is a key blue light-activated virulence factor in this microorganism. The structural basis of the light activation mechanism of this protein remains unclear. In this work, full-length LOV-HK was cloned, expressed and purified. The protein was activated by light and crystallized under a controlled illumination environment. The merge of 14 individual native data sets collected on a single crystal resulted in a complete X-ray diffraction data set to a resolution of 3.70 Å with over 2 million reflections. Crystals belong to space group P2₁2₁2₁, with unit-cell parameters a = 95.96, b = 105.30, c = 164.49 Å with a dimer in the asymmetric unit. Molecular replacement with Phaser using the individual domains as search models allowed for the reconstruction of almost the whole protein. Very recently, improved LOV-HK crystals led to a 3.25-Å resolution dataset. Refinement and model building is underway. This crystal model will represent one of the very few examples of a multi-domain histidine kinase with known structure.     

Cyclic di-GMP signalling in the virulence and environmental adaptation of Xanthomonas campestris

Cyclic di-GMP is a second messenger with a role in regulation of a range of cellular functions in diverse bacteria including the virulence of pathogens. Cellular levels of cyclic di-GMP are controlled through synthesis, catalysed by the GGDEF protein domain, and degradation by EAL or HD-GYP domains. Here we report a comprehensive study of cyclic di-GMP signalling in bacterial disease in which we examine the contribution of all proteins with GGDEF, EAL or HD-GYP domains to virulence and virulence factor production in the phytopathogen Xanthomonas campestris pathovar campestris (Xcc). Genes with significant roles in virulence to plants included those encoding proteins whose probable function is in cyclic-di-GMP synthesis as well as others (including the HD-GYP domain regulator RpfG) implicated in cyclic di-GMP degradation. Furthermore, RpfG controlled expression of a subset of these genes. A partially overlapping set of elements controlled the production of virulence factors in vitro. Other GGDEF-EAL domain proteins had no effect on virulence factor synthesis but did influence motility. These findings indicate the existence of a regulatory network that may allow Xcc to integrate information from diverse environmental inputs to modulate virulence factor synthesis as well as of cyclic di-GMP signalling systems dedicated to other specific tasks.     

Sensor domains encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase

Anthrax toxin and capsule, determinants for successful infection by Bacillus anthracis, are encoded on the virulence plasmids pXO1 and pXO2, respectively. Each of these plasmids also encodes proteins that are highly homologous to the signal sensor domain of a chromosomally encoded major sporulation sensor histidine kinase (BA2291) in this organism. B. anthracis Sterne overexpressing the plasmid pXO2-61-encoded signal sensor domain exhibited a significant decrease in sporulation that was suppressed by the deletion of the BA2291 gene. Expression of the sensor domains from the pXO1-118 and pXO2-61 genes in Bacillus subtilis strains carrying the B. anthracis sporulation sensor kinase BA2291 gene resulted in BA2291-dependent inhibition of sporulation. These results indicate that sporulation sensor kinase BA2291 is converted from an activator to an inhibitor of sporulation in its native host by the virulence plasmid-encoded signal sensor domains. We speculate that activation of these signal sensor domains contributes to the initiation of B. anthracis sporulation in the bloodstream of its infected host, a salient characteristic in the virulence of this organism, and provides an additional role for the virulence plasmids in anthrax pathogenesis.     

Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.     

Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia

AtsR is a membrane-bound hybrid sensor kinase of Burkholderia cenocepacia that negatively regulates quorum sensing and virulence factors such as biofilm production, type 6-secretion, and protease secretion. Here we elucidate the mechanism of AtsR phosphorelay by site-directed mutagenesis of predicted histidine and aspartic acid phosphoacceptor residues. We demonstrate by in vitro phosphorylation that histidine 245 and aspartic acid 536 are conserved sites of phosphorylation in AtsR, and we also identify the cytosolic response regulator AtsT (BCAM0381) as a key component of the AtsR phosphorelay pathway. Monitoring the function of AtsR and its derivatives in vivo by measuring extracellular protease activity and swarming motility confirmed the in vitro phosphorylation results. Together we find that the AtsR receiver domain plays a fine-tuning role in determining the levels of phosphotransfer from its sensor kinase domain to the AtsT response regulator.     

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.     

Immunoproteomic identification of immunogenic proteins in Cronobacter sakazakii strain BAA-894

Cronobacter spp. are emerging opportunistic pathogens. Cronobacter sakazakii is considered as the predominant species in all infections. So far, our understanding of the species’ immunogens and potential virulence factors of Cronobacter spp. remains limited. In this study, an immunoproteomic approach was used to investigate soluble and insoluble proteins from the genome-sequenced strain C. sakazakii ATCC BAA-894. Proteins were separated using two-dimensional electrophoresis, detected by Western blotting with polyclonal antibodies of C. sakazakii BAA-894, and identified using tandem mass spectrometry (MALDI-MS and MALDI-MS/MS, MS/MSMS). A total of 11 immunoreactive proteins were initially identified in C. sakazakii BAA-894, including two outer membrane proteins, four periplasmic proteins, and five cytoplasmic proteins. In silico functional analysis of the 11 identified proteins indicated three proteins that were initially described as immunogens of pathogenic bacteria. For the remaining eight proteins, one protein was categorized as a potential virulence factor involved in protection against reactive oxygen species, and seven proteins were considered to play potential roles in adhesion, invasion, and biofilm formation. To our knowledge, this is the first time that immunogenic proteins of C. sakazakii BAA-894 have been identified as immunogens and potential virulence factors by an immunoproteomics approach. Future studies should investigate the roles of these proteins in bacterial pathogenesis and modulation of host immune responses during infection to identify their potential as molecular therapeutic targets.     

Screening and identification of ClpE interaction proteins in Streptococcus pneumoniae by a bacterial two-hybrid system and co-immunoprecipitation

Hsp100/Clp proteins have crucial functions in the protein quality control, stress tolerance, and virulence of many pathogenic bacteria. ClpE is an important virulence factor involved in adherence and invasion in Streptococcus pneumoniae. To explore the underlying mechanism, we screened ClpE interaction proteins using a bacterial two-hybrid system and co-immunoprecipitation. We used ClpE as bait and constructed the pBT-ClpE bait plasmid for two-hybrid screening. Then, we constructed ClpE::GFP fusion for co-immunoprecipitation screening using anti-GFP monoclonal antibody. We obtained eight potential ClpE interaction proteins, including carbamoyl-phosphate synthase, pyruvate oxidase (SpxB), phosphoenolpyruvate-protein phosphotransferase, aminopeptidase N (pepN), L-lactate dehydrogenase, ribosomal protein S4, sensor histidine kinase (SPD_2019), and FtsW (a cell division protein). FtsW, SpxB, pepN, and SPD_2019 were confirmed to interact with ClpE using Bacterial Two-hybrid or Co-immunoprecipitation. Morphologic observations found that ΔclpE strain existed in abnormal division. β-Galactosidase activity assay suggested that ClpE contributed to the degradation of FtsW. Furthermore, FtsW could be induced by heat shock. The results suggested that ClpE might affect cell division by regulating the level of FtsW. These data may provide new insights in studying the role of ClpE in S. pneumoniae.     

The Host Plant Metabolite Glucose Is the Precursor of Diffusible Signal Factor (DSF) Family Signals in Xanthomonas campestris

Plant pathogen Xanthomonas campestris pv. campestris produces cis-11-methyl-2-dodecenoic acid (diffusible signal factor [DSF]) as a cell-cell communication signal to regulate biofilm dispersal and virulence factor production. Previous studies have demonstrated that DSF biosynthesis is dependent on the presence of RpfF, an enoyl-coenzyme A (CoA) hydratase, but the DSF synthetic mechanism and the influence of the host plant on DSF biosynthesis are still not clear. We show here that exogenous addition of host plant juice or ethanol extract to the growth medium of X. campestris pv. campestris could significantly boost DSF family signal production. It was subsequently revealed that X. campestris pv. campestris produces not only DSF but also BDSF (cis-2-dodecenoic acid) and another novel DSF family signal, which was designated DSF-II. BDSF was originally identified in Burkholderia cenocepacia to be involved in regulation of motility, biofilm formation, and virulence in B. cenocepacia. Functional analysis suggested that DSF-II plays a role equal to that of DSF in regulation of biofilm dispersion and virulence factor production in X. campestris pv. campestris. Furthermore, chromatographic separation led to identification of glucose as a specific molecule stimulating DSF family signal biosynthesis in X. campestris pv. campestris. ¹³C-labeling experiments demonstrated that glucose acts as a substrate to provide a carbon element for DSF biosynthesis. The results of this study indicate that X. campestris pv. campestris could utilize a common metabolite of the host plant to enhance DSF family signal synthesis and therefore promote virulence.     

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.     

Association of a Bacteriophage with Meningococcal Disease in Young Adults

Despite being the agent of life-threatening meningitis, Neisseria meningitidis is usually carried asymptomatically in the nasopharynx of humans and only occasionally causes disease. The genetic bases for virulence have not been entirely elucidated and the search for new virulence factors in this species is hampered by the lack of an animal model representative of the human disease. As an alternative strategy we employ a molecular epidemiological approach to establish a statistical association of a candidate virulence gene with disease in the human population. We examine the distribution of a previously-identified genetic element, a temperate bacteriophage, in 1288 meningococci isolated from cases of disease and asymptomatic carriage. The phage was over-represented in disease isolates from young adults indicating that it may contribute to invasive disease in this age group. Further statistical analysis indicated that between 20% and 45% of the pathogenic potential of the five most common disease-causing meningococcal groups was linked to the presence of the phage. In the absence of an animal model of human disease, this molecular epidemiological approach permitted the estimation of the influence of the candidate virulence factor. Such an approach is particularly valuable in the investigation of exclusively human diseases.     

Staphylococcus aureus RNAIII Binds to Two Distant Regions of coa mRNA to Arrest Translation and Promote mRNA Degradation

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.