scholarly journals High-Temperature Protein G Is an Essential Virulence Factor of Leptospira interrogans

2013 ◽  
Vol 82 (3) ◽  
pp. 1123-1131 ◽  
Author(s):  
Amy M. King ◽  
Gabriela Pretre ◽  
Thanatchaporn Bartpho ◽  
Rasana W. Sermswan ◽  
Claudia Toma ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Stress Responses ◽  
Vaccine Development ◽  
Bacterial Species ◽  
Insertion Mutant ◽  
Leptospira Interrogans ◽  
Hamster Model ◽  
Content Type ◽  
Transposon Insertion ◽  
Increased Sensitivity

ABSTRACTLeptospira interrogansis a global zoonotic pathogen and is the causative agent of leptospirosis, an endemic disease of humans and animals worldwide. There is limited understanding of leptospiral pathogenesis; therefore, further elucidation of the mechanisms involved would aid in vaccine development and the prevention of infection. HtpG (high-temperatureproteinG) is the bacterial homolog to the highly conserved molecular chaperone Hsp90 and is important in the stress responses of many bacteria. The specific role of HtpG, especially in bacterial pathogenesis, remains largely unknown. Through the use of anL. interroganshtpGtransposon insertion mutant, this study demonstrates thatL. interrogansHtpG is essential for virulence in the hamster model of acute leptospirosis. Complementation of thehtpGmutant completely restored virulence. Surprisingly, thehtpGmutant did not appear to show sensitivity to heat or oxidative stress, phenotypes common inhtpGmutants in other bacterial species. Furthermore, the mutant did not show increased sensitivity to serum complement, reduced survival within macrophages, or altered protein or lipopolysaccharide expression. The underlying cause for attenuation thus remains unknown, but HtpG is a novel leptospiral virulence factor and one of only a very small number identified to date.

scholarly journals Role of sapA and yfgA in Susceptibility to Antibody-Mediated Complement-Dependent Killing and Virulence of Salmonella enterica Serovar Typhimurium

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Edna M. Ondari ◽  
Jennifer N. Heath ◽  
Elizabeth J. Klemm ◽  
Gemma Langridge ◽  
Lars Barquist ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Protein Translocation ◽  
Invasive Disease ◽  
Immune Serum ◽  
Insertion Mutant ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Serum Killing ◽  
Increased Sensitivity

ABSTRACT The ST313 pathovar of Salmonella enterica serovar Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of S. Typhimurium. Vaccination is an attractive disease-prevention strategy, and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of S. Typhimurium to immune serum identified a repertoire of S. Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation, and metabolism. We generated defined mutant derivatives using S. Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S. Typhimurium yfgA mutant lost the characteristic Salmonella rod-shaped appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes, and demonstrated decreased ability to infect mice. In contrast, the S. Typhimurium sapA mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile and an increased ability to infect enterocytes compared with the wild type, but it had no increased ability to cause in vivo infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence and suggest the presence of different mechanisms of antibody resistance.

scholarly journals Oral Immunization with Escherichia coli Expressing a Lipidated Form of LigA Protects Hamsters against Challenge with Leptospira interrogans Serovar Copenhageni

2013 ◽  
Vol 82 (2) ◽  
pp. 893-902 ◽  
Author(s):  
Kristel Lourdault ◽  
Long-Chieh Wang ◽  
Ana Vieira ◽  
James Matsunaga ◽  
Rita Melo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Oral Vaccine ◽  
Oral Immunization ◽  
Reservoir Host ◽  
Leptospira Interrogans ◽  
Renal Tubules ◽  
Hamster Model ◽  
Content Type ◽  
E Coli ◽  
Humoral Immune

ABSTRACTLeptospirosis is a potentially fatal zoonosis transmitted by reservoir host animals that harbor leptospires in their renal tubules and shed the bacteria in their urine.Leptospira interrogansserovar Copenhageni transmitted fromRattus norvegicusto humans is the most prevalent cause of urban leptospirosis. We examinedL. interrogansLigA, domains 7 to 13 (LigA7-13), as an oral vaccine delivered byEscherichia colias a lipidated, membrane-associated protein. The efficacy of the vaccine was evaluated in a susceptible hamster model in terms of the humoral immune response and survival from leptospiral challenge. Four weeks of oral administration of liveE. coliexpressing LigA7-13 improved survival from intraperitoneal (i.p.) and intradermal (i.d.) challenge byL. interrogansserovar Copenhageni strain Fiocruz L1-130 in Golden Syrian hamsters. Immunization withE. coliexpressing LigA7-13 resulted in a systemic antibody response, and a significant LigA7-13 IgG level after the first 2 weeks of immunization was completely predictive of survival 28 days after challenge. As in previous LigA vaccine studies, all immunized hamsters that survived infection had renal leptospiral colonization and histopathological changes. In summary, an oral LigA-based vaccine improved survival from leptospiral challenge by either the i.p. or i.d. route.

scholarly journals Genes Contributing to Staphylococcus aureus Fitness in Abscess- and Infection-Related Ecologies

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Michael D. Valentino ◽  
Lucy Foulston ◽  
Ama Sadaka ◽  
Veronica N. Kos ◽  
Regis A. Villet ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
New Drugs ◽  
Insertion Mutant ◽  
Mutant Library ◽  
Entire Genome ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Transposon Insertion ◽  
Genes Encoding ◽  
Novel Targets

ABSTRACTStaphylococcus aureusis a leading cause of both community- and hospital-acquired infections that are increasingly antibiotic resistant. The emergence ofS. aureusresistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets. We generated a highly saturated transposon insertion mutant library in the genome ofS. aureusand used Tn-seq analysis to probe the entire genome, with unprecedented resolution and sensitivity, for genes of importance in infection. We further identified genes contributing to fitness in various infected compartments (blood and ocular fluids) and compared them to genes required for growth in rich medium. This resulted in the identification of 426 genes that were important forS. aureusfitness during growth in infection models, including 71 genes that could be considered essential for survival specifically during infection. These findings highlight novel as well as previously known genes encoding virulence traits and metabolic pathways important forS. aureusproliferation at sites of infection, which may represent new therapeutic targets.IMPORTANCEStaphylococcus aureuscontinues to be a leading cause of antibiotic-resistant community and nosocomial infection. With the bacterium’s acquisition of resistance to methicillin and, more recently, vancomycin, the need for the development of new drugs with novel targets is urgent. Applying a highly saturated Tn-seq mutant library to analyze fitness and growth requirements in a murine abscess and in various infection-relevant fluids, we identifiedS. aureustraits that enable it to survive and proliferate during infection. This identifies potential new targeting opportunities for the development of novel therapeutics.

scholarly journals The Vibrio cholerae Cpx Envelope Stress Response Senses and Mediates Adaptation to Low Iron

2014 ◽  
Vol 197 (2) ◽  
pp. 262-276 ◽  
Author(s):  
Nicole Acosta ◽  
Stefan Pukatzki ◽  
Tracy L. Raivio
Keyword(s):  
Vibrio Cholerae ◽  
Stress Responses ◽  
Bacterial Species ◽  
Membrane Pore ◽  
Toxic Compounds ◽  
Content Type ◽  
Major Function ◽  
Pathway Activation ◽  
Envelope Stress ◽  
Cpx Pathway

The Cpx pathway, a two-component system that employs the sensor histidine kinase CpxA and the response regulator CpxR, regulates crucial envelope stress responses across bacterial species and affects antibiotic resistance. To characterize the CpxR regulon inVibrio cholerae, the transcriptional profile of the pandemicV. choleraeEl Tor C6706 strain was examined upon overexpression ofcpxR. Our data show that the Cpx regulon ofV. choleraeis enriched in genes encoding membrane-localized and transport proteins, including a large number of genes known or predicted to be iron regulated. Activation of the Cpx pathway further led to the expression of TolC, the major outer membrane pore, and of components of two RND efflux systems inV. cholerae. We show that iron chelation, toxic compounds, or deletion of specific RND efflux components leads to Cpx pathway activation. Furthermore, mutations that eliminate the Cpx response or members of its regulon result in growth phenotypes in the presence of these inducers that, together with Cpx pathway activation, are partially suppressed by iron. Cumulatively, our results suggest that a major function of the Cpx response inV. choleraeis to mediate adaptation to envelope perturbations caused by toxic compounds and the depletion of iron.

scholarly journals Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA

2017 ◽  
Vol 55 (6) ◽  
pp. 1847-1856 ◽  
Author(s):  
Enrique Zozaya-Valdés ◽  
Jessica L. Porter ◽  
John Coventry ◽  
Janet A. M. Fyfe ◽  
Glen P. Carter ◽  
...  
Keyword(s):  
In Silico ◽  
Bacterial Species ◽  
Invasive Disease ◽  
Diagnostic Methods ◽  
Quantitative Detection ◽  
Content Type ◽  
Diagnostic Assays ◽  
Mycobacterial Genome ◽  
Increased Sensitivity

ABSTRACT Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera . Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera . We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera . Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera .

scholarly journals Genome-Wide Screening for Identification of Novel Toxin-Antitoxin Systems in Staphylococcus aureus

2019 ◽  
Vol 85 (20) ◽  
Author(s):  
Fuminori Kato ◽  
Satoshi Yoshizumi ◽  
Yoshihiro Yamaguchi ◽  
Masayori Inouye
Keyword(s):  
Staphylococcus Aureus ◽  
Genome Sequencing ◽  
Stress Responses ◽  
Noncoding Rna ◽  
Pathogenic Bacteria ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Content Type ◽  
Recent Advances ◽  
Native Host

ABSTRACT Toxin-antitoxin (TA) systems consist of toxin-inhibiting diverse cellular functions (e.g., DNA replication, transcription, and translation) and a noncoding RNA or protein antitoxin. TA systems are associated with various cellular events, such as stress responses, programmed cell death, and bacterial pathogenicity. Recent advances in genome sequencing and bioinformatics research have demonstrated that most bacteria harbor various kinds of TA modules on their chromosomes; however, there is little understanding of chromosomally encoded TA systems in the Gram-positive pathogen Staphylococcus aureus. Here, we report on newly discovered S. aureus TA systems, each of which is composed of two proteins. Manual search and gene operon prediction analysis identified eight 2-gene operons as potential candidates for TA systems. Subsequently, using an Escherichia coli host killing and rescue assay, we demonstrated that four of the eight candidates worked as TA systems, designated tsaAT, tsbAT, tscAT, and tsdAT. Moreover, the TsaT, TsbT, TscT, and TsdT toxins inhibited S. aureus growth, and the toxicity of TsbT was neutralized by coexpressing the tsbA gene in the native host, S. aureus. Further, the bioinformatics analysis of the gene clusters revealed that TsaAT, TsbAT, TscAT, and TsdAT did not exhibit sequence similarity to known bacterial TA systems, and their homologues were present only within Staphylococcus species and not among any other bacteria. Our results further advance not only the understanding of S. aureus TA systems but also the study of unannotated TA systems in various bacterial species. IMPORTANCE Recent advances in genome sequencing and bioinformatics research have demonstrated that most pathogenic bacteria harbor a large number of chromosomally encoded toxin-antitoxin (TA) modules. However, little is known about the TA systems in S. aureus. Here, we newly identified four S. aureus TA systems using a combination of manual base-by-base screening and functional analysis in E. coli. Moreover, all toxins of the identified TA systems caused growth inhibition in the native host S. aureus. Although the newly identified TA systems did not exhibit sequence similarity with known bacterial TA systems, their orthologues were conserved only among other Staphylococcus species, indicating their uniqueness to staphylococci. Our approach opens the possibility for studying unannotated TA systems in various bacterial species.

scholarly journals Screening of a Leptospira biflexa Mutant Library To Identify Genes Involved in Ethidium Bromide Tolerance

2014 ◽  
Vol 80 (19) ◽  
pp. 6091-6103 ◽  
Author(s):  
Helena Pětrošová ◽  
Mathieu Picardeau
Keyword(s):  
Antibiotic Resistance ◽  
Ethidium Bromide ◽  
Antimicrobial Agents ◽  
Genetic Manipulation ◽  
Content Type ◽  
Transposon Insertion ◽  
Damage Repair ◽  
Transposon Mutants ◽  
Living Species ◽  
Increased Sensitivity

ABSTRACTLeptospiraspp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyteL. biflexais a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants inL. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduceL. biflexasusceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n= 11), transport (n= 6), membrane structure (n= 5), stress response (n= 2), DNA damage repair (n= 1), and other processes (n= 3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth ofL. biflexain the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenicL. interrogans.

scholarly journals Nucleosome Assembly Factors CAF-1 and HIR Modulate Epigenetic Switching Frequencies in an H3K56 Acetylation-Associated Manner in Candida albicans

2013 ◽  
Vol 12 (4) ◽  
pp. 591-603 ◽  
Author(s):  
John S. Stevenson ◽  
Haoping Liu
Keyword(s):  
Cell Fate ◽  
Stress Responses ◽  
Protein Complexes ◽  
Ectopic Expression ◽  
Cell Types ◽  
Nucleosome Assembly ◽  
Content Type ◽  
Histone Gene Expression ◽  
H3k56 Acetylation ◽  
Increased Sensitivity

ABSTRACT CAF-1 and HIR are highly conserved histone chaperone protein complexes that function in the assembly of nucleosomes onto chromatin. CAF-1 is characterized as having replication-coupled nucleosome activity, whereas the HIR complex can assemble nucleosomes independent of replication. Histone H3K56 acetylation, controlled by the acetyltransferase Rtt109 and deacetylase Hst3, also plays a significant role in nucleosome assembly. In this study, we generated a set of deletion mutants to genetically characterize pathway-specific and overlapping functions of CAF-1 and HIR in C. albicans . Their roles in epigenetic maintenance of cell type were examined by using the white-opaque switching system in C. albicans . We show that CAF-1 and HIR play conserved roles in UV radiation recovery, repression of histone gene expression, correct chromosome segregation, and stress responses. Unique to C. albicans , the cac2Δ/Δ mutant shows increased sensitivity to the Hst3 inhibitor nicotinamide, while the rtt109Δ/Δ cac2Δ/Δ and hir1Δ/Δ cac2Δ/Δ mutants are resistant to nicotinamide. CAF-1 plays a major role in maintaining cell types, as the cac2Δ/Δ mutant exhibited increased switching frequencies in both directions and switched at a high frequency to opaque in response to nicotinamide. Like the rtt109Δ/Δ mutant, the hir1Δ/Δ cac2Δ/Δ double mutant is defective in maintaining the opaque cell fate and blocks nicotinamide-induced opaque formation, and the defects are suppressed by ectopic expression of the master white-opaque regulator Wor1. Our data suggest an overlapping function of CAF-1 and HIR in epigenetic regulation of cell fate determination in an H3K56 acetylation-associated manner.

scholarly journals Plasmid-Encoded MCP Is Involved in Virulence, Motility, and Biofilm Formation of Cronobacter sakazakii ATCC 29544

2014 ◽  
Vol 83 (1) ◽  
pp. 197-204 ◽  
Author(s):  
Younho Choi ◽  
Seongok Kim ◽  
Hyelyeon Hwang ◽  
Kwang-Pyo Kim ◽  
Dong-Hyun Kang ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Protein Gene ◽  
Open Reading Frames ◽  
Insertion Mutant ◽  
Biological Processes ◽  
Cronobacter Sakazakii ◽  
Content Type ◽  
Rat Pups ◽  
Transposon Insertion ◽  
Reading Frames

The aim of this study was to elucidate the function of the plasmid-bornemcp(methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles inCronobacter sakazakiiATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, inC. sakazakiiATCC 29544. Anin silicoanalysis of pCSA2 revealed that it included six putative open reading frames, and one of them wasmcp. Themcpmutant was defective for invasion into and adhesion to epithelial cells, and the virulence of themcpmutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility ofC. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functionalmcpgene. Furthermore, a lack of themcpgene also impaired the ability ofC. sakazakiito form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence ofC. sakazakiiATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in theC. sakazakiisequence type 8 (ST8) lineage.

scholarly journals FlaA Proteins in Leptospira interrogans Are Essential for Motility and Virulence but Are Not Required for Formation of the Flagellum Sheath

2012 ◽  
Vol 80 (6) ◽  
pp. 2019-2025 ◽  
Author(s):  
Ambroise Lambert ◽  
Mathieu Picardeau ◽  
David A. Haake ◽  
Rasana W. Sermswan ◽  
Amporn Srikram ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Animal Models ◽  
Acute Infection ◽  
Transposon Mutagenesis ◽  
Leptospira Interrogans ◽  
Wild Type ◽  
Hamster Model ◽  
Normal Morphology ◽  
Content Type ◽  
Periplasmic Flagella

ABSTRACTSpirochetes have periplasmic flagella composed of a core surrounded by a sheath. The pathogenLeptospira interroganshas fourflaB(proposed core subunit) and twoflaA(proposed sheath subunit) genes. TheflaAgenes are organized in a locus withflaA2immediately upstream offlaA1. In this study,flaA1andflaA2mutants were constructed by transposon mutagenesis. Both mutants still produced periplasmic flagella. TheflaA1mutant did not produce FlaA1 but continued to produce FlaA2 and retained normal morphology and virulence in a hamster model of infection but had reduced motility. TheflaA2mutant did not produce either the FlaA1 or the FlaA2 protein. Cells of theflaA2mutant lacked the distinctive hook-shaped ends associated withL. interrogansand lacked translational motility in liquid and semisolid media. These observations were confirmed with a second, independentflaA2mutant. TheflaA2mutant failed to cause disease in animal models of acute infection. Despite lacking FlaA proteins, the flagella of theflaA2mutant were of the same thickness as wild-type flagella, as measured by electron microscopy, and exhibited a normal flagellum sheath, indicating that FlaA proteins are not essential for the synthesis of the flagellum sheath, as observed for other spirochetes. This study shows that FlaA subunits contribute to leptospiral translational motility, cellular shape, and virulence.

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