scholarly journals Defining the core essential genome ofPseudomonas aeruginosa

2019 ◽  
Vol 116 (20) ◽  
pp. 10072-10080 ◽  
Author(s):  
Bradley E. Poulsen ◽  
Rui Yang ◽  
Anne E. Clatworthy ◽  
Tiantian White ◽  
Sarah J. Osmulski ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Growth Conditions ◽  
Essential Genes ◽  
The Core ◽  
Transposon Insertion ◽  
Wide Range ◽  
Transposon Insertion Sequencing ◽  
Antibiotic Discovery

Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. While numerous factors contributed to the disappointing yield, one factor was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both the genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains ofPseudomonas aeruginosaon five different media and developed a statistical model,FiTnEss, to classify genes as essential versus nonessential across all strain–medium combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of four strains was typically sufficient inP. aeruginosato converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.

scholarly journals Defining the core essential genome ofPseudomonas aeruginosa

2018 ◽  
Author(s):  
Bradley E. Poulsen ◽  
Rui Yang ◽  
Anne E. Clatworthy ◽  
Tiantian White ◽  
Sarah J. Osmulski ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Growth Conditions ◽  
Essential Genes ◽  
The Core ◽  
Transposon Insertion ◽  
Wide Range ◽  
Transposon Insertion Sequencing ◽  
Antibiotic Discovery

Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. It is becoming clear that a major limitation was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number ofin vitrolaboratory conditions. In fact, the essentiality of a gene can depend on both genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains ofPseudomonas aeruginosaon five different media and developed a novel statistical model,FiTnEss, to classify genes as essential versus non-essential across all strain-media combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of 4 strains was typically sufficient inP. aeruginosato converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant toin vivoinfection, and thus to represent attractive targets for novel drug discovery.

scholarly journals Phage Therapy as a Focused Management Strategy in Aquaculture

2021 ◽  
Vol 22 (19) ◽  
pp. 10436
Author(s):  
José Ramos-Vivas ◽  
Joshua Superio ◽  
Jorge Galindo-Villegas ◽  
Félix Acosta
Keyword(s):  
Bacterial Infections ◽  
Therapeutic Potential ◽  
Current Knowledge ◽  
Control Strategies ◽  
Bacterial Species ◽  
Lytic Enzymes ◽  
Wide Range ◽  
Cultured Fish

Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to antibiotics in the management of bacterial infections of a wide range of organisms including cultured fish. Their natural immunogenicity often induces the modulation of a variated collection of immune responses within several types of immunocytes while promoting specific mechanisms of bacterial clearance. However, to achieve standardized treatments at the practical level and avoid possible side effects in cultivated fish, several improvements in the understanding of their biology and the associated genomes are required. Interestingly, a particular feature with therapeutic potential among all phages is the production of lytic enzymes. The use of such enzymes against human and livestock pathogens has already provided in vitro and in vivo promissory results. So far, the best-understood phages utilized to fight against either Gram-negative or Gram-positive bacterial species in fish culture are mainly restricted to the Myoviridae and Podoviridae, and the Siphoviridae, respectively. However, the current functional use of phages against bacterial pathogens of cultured fish is still in its infancy. Based on the available data, in this review, we summarize the current knowledge about phage, identify gaps, and provide insights into the possible bacterial control strategies they might represent for managing aquaculture-related bacterial diseases.

scholarly journals In VivoExpression of Streptococcus pyogenes Immunogenic Proteins during Tibial Foreign Body Infection

2014 ◽  
Vol 82 (9) ◽  
pp. 3891-3899 ◽  
Author(s):  
Jeffrey A. Freiberg ◽  
Kevin S. McIver ◽  
Mark E. Shirtliff
Keyword(s):  
Group A Streptococcus ◽  
Rabbit Model ◽  
Growth Conditions ◽  
Rabbit Serum ◽  
Tissue Samples ◽  
Wide Range ◽  
Group A ◽  
Immunogenic Proteins

ABSTRACTGroup A streptococcus (GAS) is an important human pathogen that causes a number of diseases with a wide range of severities. While all known strains of GAS are still sensitive to penicillin, there have been reports of antibiotic treatment failure in as many as 20% to 40% of cases. Biofilm formation has been implicated as a possible cause for these failures. A biofilm is a microbially derived, sessile community where cells grow attached to a surface or as a bacterial conglomerate and surrounded by a complex extracellular matrix. While the ability of group A streptococcus to form biofilms in the laboratory has been shown, there is a lack of understanding of the role of GAS biofilms during an infection. We hypothesized that during infections, GAS exhibits a biofilm phenotype, complete with unique protein expression. To test this hypothesis, a rabbit model of GAS osteomyelitis was developed. A rabbit was inoculated with GAS using an infected indwelling device. Following the infection, blood and tissue samples were collected. Histological samples of the infected tibia were prepared, and the formation of a biofilmin vivowas visualized using peptide nucleic acid fluorescentin situhybridization (PNA-FISH) and confocal microscopy. In addition, Western blotting with convalescent rabbit serum detected cell wall proteins expressedin vitrounder biofilm and planktonic growth conditions. Immunogenic proteins were then identified using matrix-assisted laser desorption ionization–time of flight tandem mass spectrometry (MALDI-TOF/TOF MS). These identities, along with thein vivoresults, support the hypothesis that GAS forms biofilms during an infection. This unique phenotype should be taken into consideration when designing a vaccine or any other treatment for group A streptococcus infections.

scholarly journals Molecular Cloning and Analysis of a Putative Siderophore ABC Transporter from Staphylococcus aureus

2000 ◽  
Vol 68 (11) ◽  
pp. 6281-6288 ◽  
Author(s):  
Julie A. Morrissey ◽  
Alan Cockayne ◽  
Philip J. Hill ◽  
Paul Williams
Keyword(s):  
Staphylococcus Aureus ◽  
Abc Transporter ◽  
Growth Conditions ◽  
Expression Library ◽  
Phase Partitioning ◽  
Wide Range ◽  
Monospecific Antisera ◽  
Triton X

ABSTRACT From a mass-excised Staphylococcus aureus λZapII expression library, we cloned an operon encoding a novel ABC transporter with significant homology to bacterial siderophore transporter systems. The operon encodes four genes designatedsstA, -B, -C, and -Dencoding two putative cytoplasmic membrane proteins (sstAand sstB), an ATPase (sstC), and a membrane-bound 38-kDa lipoprotein (sstD). Thesst operon is preceded by two putative Fur boxes, which indicated that expression of the sst operon was likely to be iron dependent. SstD was overexpressed inEscherichia coli, purified by Triton X-114 phase partitioning, and used to generate monospecific antisera in rats. Immunoblotting studies located SstD in the membrane fraction ofS. aureus and showed that expression of the lipoprotein was reduced under iron-rich growth conditions. Triton X-114 partitioning studies on isolated membranes provided additional biochemical evidence that SstD in S. aureus is a lipoprotein. Immunoreactive polypeptides of approximately 38 kDa were detected in a wide range of staphylococcal species, but no antigenic homolog was detected inBacillus subtilis. Expression of SstD in vivo was confirmed by immunoblotting studies with S. aureus recovered from a rat intraperitoneal chamber implant model. To further define the contribution of SstD in promoting growth of S. aureus in vitro and in vivo, we used antisense RNA technology to modulate expression of SstD. Expression of antisense sstD RNA inS. aureus resulted in a decrease in SstD expression under both iron-rich and iron-restricted growth conditions. However, this reduction in SstD levels did not affect the growth of S. aureus in vitro in an iron-limited growth medium or when grown in an intraperitoneal rat chamber implant model in vivo.

scholarly journals Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

2019 ◽  
Author(s):  
Alyson R. Warr ◽  
Troy P. Hubbard ◽  
Diana Munera ◽  
Carlos J. Blondel ◽  
Pia Abel zur Wiesch ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Intestinal Colonization ◽  
E Coli ◽  
Rabbit Colon ◽  
Transposon Insertion ◽  
Food Borne ◽  
K 12 ◽  
Transposon Insertion Sequencing

AbstractEnterohemorrhagicEscherichia coliO157:H7 (EHEC) is an important food-borne pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to identify genes required for EHEC and commensalE. coliK-12 growth in vitro and for EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci contribute to EHEC’s but not to K-12’s growth in vitro, suggesting that gene acquisition during EHEC evolution has heightened the pathogen’s reliance on certain metabolic processes that are dispensable for K-12. There was a restrictive bottleneck for EHEC colonization of the rabbit colon, which complicated identification of EHEC genes facilitating growth in vivo. Both a refined version of an existing analytic framework as well as PCA-based analysis were used to compensate for the effects of the infection bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion apparatus is required for growth in vivo and revealed that only a few effectors are critical for in vivo fitness. Numerous mutants not previously associated with EHEC survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative in vivo fitness factors were validated. Some were found to contribute to efficient type-three secretion while others, includingtatABC, oxyR, envC, acrAB, andcvpA, promote EHEC resistance to host-derived stresses encountered in vivo.cvpA, which is also required for intestinal growth of several other enteric pathogens, proved to be required for EHEC,Vibrio choleraeandVibrio parahaemolyticusresistance to the bile salt deoxycholate. Collectively, our findings provide a comprehensive framework for understanding EHEC growth in the intestine.Author SummaryEnterohemorrhagicE. coli(EHEC) are important food-borne pathogens that infect the colon. We created a highly saturated EHEC transposon library and used transposon insertion sequencing to identify the genes required for EHEC growth in vitro and in vivo in the infant rabbit colon. We found that there is a large infection bottleneck in the rabbit model of intestinal colonization, and refined two analytic approaches to facilitate rigorous identification of new EHEC genes that promote fitness in vivo. Besides the known type III secretion system, more than 200 additional genes were found to contribute to EHEC survival and/or growth within the intestine. The requirement for some of these new in vivo fitness factors was confirmed, and their contributions to infection were investigated. This set of genes should be of considerable value for future studies elucidating the processes that enable the pathogen to proliferate in vivo and for design of new therapeutics.

scholarly journals Genetic analysis of Vibrio parahaemolyticus intestinal colonization

2016 ◽  
Vol 113 (22) ◽  
pp. 6283-6288 ◽  
Author(s):  
Troy P. Hubbard ◽  
Michael C. Chao ◽  
Sören Abel ◽  
Carlos J. Blondel ◽  
Pia Abel zur Wiesch ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Vibrio Parahaemolyticus ◽  
Human Pathogens ◽  
Transposon Insertion ◽  
Host Infection ◽  
Transposon Insertion Sequencing ◽  
Genetic Contributions ◽  
Mammalian Intestine

Vibrio parahaemolyticus is the most common cause of seafood-borne gastroenteritis worldwide and a blight on global aquaculture. This organism requires a horizontally acquired type III secretion system (T3SS2) to infect the small intestine, but knowledge of additional factors that underlie V. parahaemolyticus pathogenicity is limited. We used transposon-insertion sequencing to screen for genes that contribute to viability of V. parahaemolyticus in vitro and in the mammalian intestine. Our analysis enumerated and controlled for the host infection bottleneck, enabling robust assessment of genetic contributions to in vivo fitness. We identified genes that contribute to V. parahaemolyticus colonization of the intestine independent of known virulence mechanisms in addition to uncharacterized components of T3SS2. Our study revealed that toxR, an ancestral locus in Vibrio species, is required for V. parahaemolyticus fitness in vivo and for induction of T3SS2 gene expression. The regulatory mechanism by which V. parahaemolyticus ToxR activates expression of T3SS2 resembles Vibrio cholerae ToxR regulation of distinct virulence elements acquired via lateral gene transfer. Thus, disparate horizontally acquired virulence systems have been placed under the control of this ancestral transcription factor across independently evolved human pathogens.

scholarly journals Array-assisted Characterization of a Fucosyltransferase Required for the Biosynthesis of Complex Core Modifications of Nematode N-Glycans

2013 ◽  
Vol 288 (29) ◽  
pp. 21015-21028 ◽  
Author(s):  
Shi Yan ◽  
Sonia Serna ◽  
Niels-Christian Reichardt ◽  
Katharina Paschinger ◽  
Iain B. H. Wilson
Keyword(s):  
Cell Surfaces ◽  
C Elegans ◽  
The Core ◽  
Wide Range ◽  
Biological Recognition ◽  
Complex Core ◽  
Recurrent Theme

Fucose is a common monosaccharide component of cell surfaces and is involved in many biological recognition events. Therefore, definition and exploitation of the specificity of the enzymes (fucosyltransferases) involved in fucosylation is a recurrent theme in modern glycosciences. Despite various studies, the specificities of many fucosyltransferases are still unknown, so new approaches are required to study these. The model nematode Caenorhabditis elegans expresses a wide range of fucosylated glycans, including N-linked oligosaccharides with unusual complex core modifications. Up to three fucose residues can be present on the standard N,N′-diacetylchitobiose unit of these N-glycans, but only the fucosyltransferases responsible for transfer of two of these (the core α1,3-fucosyltransferase FUT-1 and the core α1,6-fucosyltransferase FUT-8) were previously characterized. By use of a glycan library in both array and solution formats, we were able to reveal that FUT-6, another C. elegans α1,3-fucosyltransferase, modifies nematode glycan cores, specifically the distal N-acetylglucosamine residue; this result is in accordance with glycomic analysis of fut-6 mutant worms. This core-modifying activity of FUT-6 in vitro and in vivo is in addition to its previously determined ability to synthesize Lewis X epitopes in vitro. A larger scale synthesis of a nematode N-glycan core in vitro using all three fucosyltransferases was performed, and the nature of the glycosidic linkages was determined by NMR. FUT-6 is probably the first eukaryotic glycosyltransferase whose specificity has been redefined with the aid of glycan microarrays and so is a paradigm for the study of other unusual glycosidic linkages in model and parasitic organisms.

scholarly journals The Molecular Basis of FimT-mediated DNA Uptake during Bacterial Natural Transformation

2021 ◽  
Author(s):  
Sebastian A.G. Braus ◽  
Francesca L. Short ◽  
Stefanie Holz ◽  
Matthew J.M. Stedman ◽  
Alvar D. Gossert ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Molecular Basis ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Wide Range

AbstractNaturally competent bacteria encode sophisticated protein machineries for the uptake and translocation of exogenous DNA into the cell. If this DNA is integrated into the bacterial genome, the bacterium is said to be naturally transformed. Most competent bacterial species utilise type IV pili for the initial DNA uptake step. These proteinaceous cell-surface structures are composed of thousands of pilus subunits (pilins), designated as major or minor according to their relative abundance in the pilus. In this study, we show that the minor pilin FimT plays an important role in the natural transformation of Legionella pneumophila. We used NMR spectroscopy, in vitro DNA binding assays and in vivo transformation assays to understand the molecular basis of FimT’s role in this process. FimT directly interacts with DNA via an electropositive patch, rich in arginines, several of which are well-conserved and located in FimT’s conformationally flexible C-terminal tail. We also show that FimT orthologues from other γ-Proteobacteria share the ability to bind to DNA. Our functional characterisation and comprehensive bioinformatic analysis of FimT, suggest that it plays an important role for DNA uptake in a wide range of competent species.

scholarly journals Magnesium-Dependent Promotion of H2O2 Production Increases Ecological Competitiveness of Oral Commensal Streptococci

2020 ◽  
Vol 99 (7) ◽  
pp. 847-854
Author(s):  
X. Cheng ◽  
S. Redanz ◽  
P. Treerat ◽  
H. Qin ◽  
D. Choi ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Bacterial Species ◽  
Growth Conditions ◽  
H2o2 Production ◽  
Potential Mechanism ◽  
Biophotonic Imaging ◽  
Oral Colonization ◽  
Low Passage

The pyruvate oxidase (SpxB)–dependent production of H2O2 is widely distributed among oral commensal streptococci. Several studies confirmed the ability of H2O2 to antagonize susceptible oral bacterial species, including caries-associated Streptococcus mutans as well as several periodontal pathobionts. Here we report a potential mechanism to bolster oral commensal streptococcal H2O2 production by magnesium (Mg2+) supplementation. Magnesium is a cofactor for SpxB catalytic activity, and supplementation increases the production of H2O2 in vitro. We demonstrate that Mg2+ affects spxB transcription and SpxB abundance in Streptococcus sanguinis and Streptococcus gordonii. The competitiveness of low-passage commensal streptococcal clinical isolates is positively influenced in antagonism assays against S. mutans. In growth conditions normally selective for S. mutans, Mg2+ supplementation is able to increase the abundance of S. sanguinis in dual-species biofilms. Using an in vivo biophotonic imaging platform, we further demonstrate that dietary Mg2+ supplementation significantly improves S. gordonii oral colonization in mice. In summary, our results support a role for Mg2+ supplementation as a potential prebiotic to promote establishment of oral health–associated commensal streptococci.

scholarly journals A first look at the essential genes of Pseudomonas protegens

2021 ◽  
Author(s):  
Bradley E. Poulsen
Keyword(s):  
Bacterial Species ◽  
Essential Genes ◽  
Sequencing Data ◽  
Comparative Analyses ◽  
Transposon Insertion ◽  
Commensal Species ◽  
Transposon Insertion Sequencing

Transposon insertion sequencing is a useful tool to identify the genes that are essential for a bacterial species to grow and divide effectively. In this issue of Journal of Bacteriology, Fabian et al. present the first set of transposon insertion sequencing data highlighting the genes essential to the plant-commensal species Pseudomonas protegens strain Pf-5 and perform comparative analyses with other Pseudomonads.

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